Compounds Useful for Inhibiting Metastasis from Cancer and Methods Using Same

ABSTRACT

The present invention includes compositions that are useful in preventing or treating metastasis in a subject diagnosed with cancer. The present invention also includes methods of preventing or treating metastasis in a subject diagnosed with cancer, wherein the method comprises administering to the subject in need thereof an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one CX 3 CR1 or fractalkine antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of, and claimspriority to, U.S. patent application Ser. No. 15/174,227, filed Jun. 6,2016, now allowed, which is a continuation of, and claims priority to,U.S. patent application Ser. No. 13/776,220, filed Feb. 25, 2013, nowissued as U.S. Pat. No. 9,375,474, which is a continuation of, andclaims priority to, U.S. patent application Ser. No. 13/312,540, filedDec. 6, 2011, now issued as U.S. Pat. No. 8,435,993, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.61/420,640, filed Dec. 7, 2010, all of which applications are herebyincorporated by reference in their entireties herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberW81XWH-09-1-0593 awarded by the Department of Defense/US Army (BreastCancer Program) and under grant number DA015014 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Metastasis or metastatic disease is the spread of a disease from oneorgan or part to another non-adjacent organ or part. Metastatic diseaseis primarily but not uniquely associated with malignant tumor cells andinfections (Klein, 2008, Science 321(5897):1785-88; Chiang & Massagué,2008, New Engl. J. Med. 359(26):2814-23).

Cancer occurs after a single cell in a tissue is genetically damaged inways that result in the formation of a putative cancer stem cellpossessing a malignant phenotype. These cancer stem cells are able toundergo uncontrolled abnormal mitosis, which serves to increase thetotal number of cancer cells at that location. When the area of cancercells at the originating site become clinically detectable, it is calledprimary tumor. Some cancer cells also acquire the ability to penetrateand infiltrate surrounding normal tissues in the local area, forming anew tumor. The newly formed tumor in the adjacent site within the tissueis called a local metastasis.

Some cancer cells acquire the ability to penetrate the walls oflymphatic and/or blood vessels, after which they are able to circulatethrough the bloodstream (circulating tumor cells) to other sites andtissues in the body. This process is known (respectively) as lymphaticor hematogenous spread. After the tumor cells come to rest at anothersite, they re-penetrate through the vessel or walls (extravasion),continue to multiply, and eventually another clinically detectable tumoris formed. This new tumor is known as a metastatic (or secondary) tumor.Metastasis is one of the hallmarks of malignancy. Most tumors and otherneoplasms can metastasize, although in varying degrees (e.g. basal cellcarcinoma rarely metastasize) (Kumar et al., 2005,“Robbins and CotranPathologic Basis of Disease”, 7th ed., Philadelphia: Elsevier Saunders).

Metastatic tumors are very common in the late stages of cancer. The mostcommon places for the metastases to occur are the lungs, liver, brain,and the bones. There is also a propensity for certain tumors to seed inparticular organs. For example, prostate cancer usually metastasizes tothe bones. In a similar manner, colon cancer has a tendency tometastasize to the liver. Stomach cancer often metastasizes to the ovaryin women. Breast tumor cells often metastasize to bone tissue. Studieshave suggested that these tissue-selective metastasis processes are dueto specific anatomic and mechanical routes.

Currently, only six percent of women that are first diagnosed withbreast adenocarcinoma present with metastases (Hortobagyi et al.,“Neoplasm of the breast”. In: Cancer Medicine. B C Decker; Holland-Frei,Ed., 2006, p.1584-643). Unfortunately, between twenty and fifty percentof them will eventually develop metastatic disease (Hortobagyi et al.,“Neoplasm of the breast”. In: Cancer Medicine. B C Decker; Holland-Frei,Ed., 2006, p.1584-643) Metastases are responsible for an intolerablyhigh number of deaths among patients that would otherwise be almostinvariably cured by surgical resection and adjuvant therapy (Lu et al.,2009, Cancer Res. 69:4951-53). Autopsy studies have estimated that 70%of advanced breast cancer patients have skeletal metastases (Bussard etal., 2008, Cancer Met. Rev. 27:41-55). These secondary bone tumors causesignificant morbidity, leading to considerable pain, spinal cordcompression and pathological fractures (Coleman, 1997, Cancer80:1588-94). In addition, when breast cancer cells have disseminated tothe skeleton, the resulting bone tumors can be treated only withpalliative measures (Body & Mancini, 2002, Off. J. Multi-Natl. Assoc.Supp. Care Cancer 10:399-407; Coleman et al., 2008, Clin. Cancer Res.14:6387-95; Costa & Major, 2009, Nat. Clin. Pract. Oncol. 6:163-74). Themajority of patients develop metastases years after initial treatment ofthe primary breast tumor. The appearance of late metastases can indeedbe attributed to cancer cells disseminated to secondary tissues duringdifferent stages of primary tumor progression and remained dormant forvariable periods of time. In fact, both early dissemination and dormancyof tumor cells are supported by strong evidence (Huseman et al., 2008,Cancer Cell 13:58-68; Aguirre-Ghiso, 2007, Nat. Rev. Cancer 7:834-46).

Due to the limited size of breast tumors that are first diagnosed today,the vast majority of patients are considered to be viable candidates forbreast-conserving surgery (BCS) or lumpectomy. Since BCS minimizes thephysical and psychological impact of breast surgery, this approach iswidely preferred by patients (Veronesi et al., 2005, Lancet, 365:17271;Morrow, 2009, BMJ 338:b557). In addition, based on studies that reportedcomparable survival rates between lumpectomy and more radical approachessuch as mastectomy, oncologic surgeons are also in favor of this form oftreatment (Veronesi et al., 2002, N. Engl. J. Med. 347:1227; Fisher etal., 2002, N. Engl. J. Med. 347:1233). However, the conclusions of thesestudies are being challenged by a meta-analysis showing that for everyfour local recurrences prevented, one breast cancer death could beavoided (Clarke et al., 2005, Lancet, 366:2087). In fact, followinglumpectomy only 37% of breasts are found tumor-free and between 22% and59% of patients will need re-intervention because positive or closemargins are detected (Sabel et al., 2009, J. Surg. Oncol. 99:99). Inaddition, reexcision or adjuvant therapies, such as local irradiation orchemotherapy, are normally started several weeks or even months afterprimary surgery (Buchholz, 2009, N. Engl. J. Med. 360:63; Balduzzi etal., 2010, Cancer Treat. Rev. 36:443) to allow for complete patient'srecovery and improve post-operatory aesthetic results.

However, the stroma at the site of tumor removal is characterized byaltered angiogenesis, immune cells infiltration and activation ofcancer-associated fibroblasts (Hofer et al.,“Wound-induced tumorprogression: a probable role in recurrence after tumor resection,”Archives of Surgery (Chicago, Ill: 1960), 133, 383, 1998; Stuelten etal., 2008, Cancer Res. 68:7278). These events are potentially able topromote perioperative proliferation and motility of residual cancercells, thereby allowing their escape into the circulation (Coffey etal., 2006, BioEssays, 28:433; Coffey et al., 2003, Lancet Oncol. 4:760).Even in the presence of dormant cancer cells already lodged into distantsites, the additional spreading of these cells would produce new wavesof micrometastases.

The arrest of circulating cancer cells to the skeleton is highlydependent on specific adhesive interactions with the endothelial cellslining the marrow sinusoids (Lehr & Pienta, 1998, J. Natl. Cancer Inst.90:118-23; Scott et al., 2001, Br. J. Cancer 84:1417; Glinskii et al,2005, Neoplasia 7:522-27). The required next step is the extravasationof adherent cancer cells drawn by chemo attractant cues generated by thesurrounding stroma (Liotta, 2001, Nature 410:24-25). The similaritiesbetween cancer cell dissemination and leukocyte trafficking lead to theidentification of chemokines as crucial players in both sets of events(Mantovani et al., 2010, Cyt. & Growth Factor Rev. 21:27-39).

The interactions between the chemokine CXCL12 (SDF-1) and its receptorCXCR4 have been extensively studied (Muller et al., 2001,Nature410:50-56; Dewan et al., 2006, Biomed Pharmacother. 60:273-76).The role of CXCR4 in cell adhesion appears to be dependent on thesecondary induction of αvβ3 integrin presentation on the surface ofcancer cells and consequent binding to vascular adhesion molecules (Englet al., 2006, Neoplasia 8:290-301). However, CXCR4 inhibition did notblock the binding of colon cancer cells to the liver endothelium, butdid limit extravasation (Gassman et al., 2009, Neoplasia 11:651-61).Thus, similarly to its role in hematopoietic stem cells homing, thesoluble chemokine CXCL12 seems to be an important player in cancer cellmigration into the bone microenvironment rather than mediating adhesiveinteractions with CXCR4-bearing cells (Gassman, 2008, Clin. Exp.Metastasis 25:171-81).

CX3C chemokine receptor 1 (CX₃CR1), also known as the fractalkinereceptor or G-protein coupled receptor 13 (GPR13), is a protein that inhumans is encoded by the CX₃CR1 gene (Robertson, 2002, J. Leukoc. Biol.71(2):173-83; Raport et al., 1995, Gene 163(2):295-99). This receptorbinds the chemokine CX₃CL1 (also called neurotactin or fractalkine orFKN). FKN is a transmembrane protein that is cleaved into a solublemolecule with potent chemoattractant properties (Bazan et al., 1997,Nature 385:640-44). In its membrane-bound form, FKN can establish strongand stable adhesive interactions with its receptor CX₃CR1, and does notrequire any downstream signaling to induce activation of additionaladhesion molecules (Haskell et al., 1999, J. Biol. Chem. 274:10053-58;Imai et al., 1997, Cell. 91:521-30; Goda et al., 2000, J. Immun.64:4313). Prostate cancer cells were shown to express CX₃CR1 and, underdynamic-flow conditions, to adhere to human bone marrow endothelialcells in a FKN-dependent manner (Shulby et al., 2004, Cancer Res.64:4693-98). In addition, CX₃CR1 was shown to be expressed in a highpercentage of prostate cancer tissues while human bone marrowsupernatants contain soluble FKN, which is released from cells of thebone stroma through a mechanism regulated by androgens (Jamieson et al.,2008, Cancer Res. 68:1715-22). A role for the FKN/CX₃CR1 pair inmetastasis is also supported by the observation that there is acorrelation between CX₃CR1 expression in primary breast tumors andclinical metastases. In addition, CX₃CR1 expression in pancreatic tumorcells was found to promote the infiltration of the central nervoussystem (Marchesi et al., 2008, Cancer Res. 68:9060-69). Finally, FKN andCX₃CR1 have been recently reported to be involved in adhesion ofneuroblastoma cells to the bone in an in vitro system (Nevo et al.,2009, Cancer Lett. 273:127-39).

CX₃CR1 was previously detected in the epithelial compartment of normaland malignant human prostate gland tissues (Shulby et al., 2004, CancerRes. 64:4693; FIG. 1). FKN was also detected both in the solublefraction of human bone marrow (˜2 ng/ml) and on the surface of humanbone marrow endothelial cells (Jamieson et al., 2008, Cancer Res.68:1715). Human tissue microarrays containing 172 samples of breastcancer were examined, and 131 were found to be positive for CX₃CR1expression (FIG. 2). Because of the high propensity shown by severalsolid tumors, including breast cancer, to target the skeleton, theseresults support the idea that functional interactions between FKN in thebone and CX₃CR1 on cancer cells are involved in skeletal metastasis.This model was strengthened by the detection of CX₃CR1 also in severalhuman breast cancer cell lines; the superior ability of MDA-231 cells toarrest at the skeleton through the blood circulation of mice was relatedto CX₃CR1 expression as compared to other cell lines such as MDA-436that fail to express this receptor (FIG. 3).

Despite the extensive research on the mechanisms of cancer metastasis,there is not a validated and effective approach to minimize thedevelopment of metastasis in patients afflicted with primary tumors.There is a need in the art to identify a method of treatment thatefficiently avoids, delays or minimizes the development of metastatictumors in patients, especially in the context of metastatic bone cancerassociated with primary prostate or breast cancers. The presentinvention fulfills these needs.

BRIEF SUMMARY OF THE INVENTION

The invention includes a composition comprising at least one agentselected from the group consisting of:

-   (i) a compound of Formula (I):

wherein in (I):

A¹ is CH₂ or cyclopentane-1,3-diyl;

n is 0, 1, 2, 3, 4 or 5 if A is CH₂, or n is 0, 1, or 2 if A iscyclopentane-1,3-diyl;

A² and A³ are both H, or A² and A³ combine to form a divalent radicalselected from the group consisting of methanediyl, ethane-1,2-diyl, orpropane-1,3-diyl;

A⁴ is CH₂, CH(CF₃), or CF₂;

R¹ and R² are both H, and R³ is selected from the group consisting of:

wherein ring A is selected from the group consisting of phenyl,substituted phenyl, pyridinyl, substituted pyridinyl, thiophenyl,substituted thiophenyl, 1H-pyrazole, and 1-(C₁-C₆) alkyl-1H-pyrazole;

R⁴ is nil or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl, compound ofFormula (I) is a quaternary ammonium salt;

R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl, substitutedphenyl, heteroaryl, substituted heteroaryl;

R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or substitutedaryl; and,

R⁷ is OH, CH₂OH, or C(═O)OR⁶;

-   (ii) a compound of Formula (IX):

R^(A)—R^(B)   (IX), wherein:

R^(A) is a group selected from the group consisting of:

R^(B) is a group selected from the group consisting of:

-   (iii) a compound of Formula (X):

wherein R¹ is (C₁-C₈) alkyl, substituted (C₁-C₈) alkyl, phenyl,substituted phenyl, (C₁-C₈) cycloalkyl or substituted (C₁-C₈)cycloalkyl;

-   (iv) a compound of Formula (XI):

wherein

R is selected from the group consisting of:

each occurrence of R¹ and R⁴ is independently H, (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,

R² and R³ are independently H, (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,phenyl, substituted phenyl, (C₁-C₆) cycloalkyl, substituted (C₁-C₆)cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or NHAc;

-   (v) a compound of Formula (XII):

wherein

R is selected from the group consisting of:

each occurrence of R¹, R⁴ and R⁵ is independently H, (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,

R² and R³ are independently H, (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,phenyl, substituted phenyl, (C₁-C₆) cycloalkyl, substituted (C₁-C₆)cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or NHAc;

-   (vi) a CX₃CR1 or fractalkine antibody; and    combinations thereof, and a pharmaceutically acceptable salt    thereof.

In one embodiment, the at least one agent is selected from the groupconsisting of:5-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one;5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one; combinationsthereof, and a salt thereof. In another embodiment, the compositionfurther comprises at least one pharmaceutically acceptable carrier. Inyet another embodiment, the antibody comprises an antibody selected froma polyclonal antibody, a monoclonal antibody, a humanized antibody, asynthetic antibody, a heavy chain antibody, a human antibody, abiologically active fragment of an antibody, and combinations thereof.

The invention also includes a method of preventing or treatingmetastasis in a subject diagnosed with cancer. The method comprisesadministering to the subject in need thereof an effective amount of apharmaceutical formulation comprising at least one pharmaceuticallyacceptable carrier and at least one CX₃CR1 or fractalkine antagonist.

In one embodiment, the subject is subjected to primary surgery relatedto the cancer. In another embodiment, administering the pharmaceuticalformulation takes place before, during or after the primary surgery. Inyet another embodiment, the cancer comprises a solid cancer. In yetanother embodiment, the solid cancer is selected from the groupconsisting of breast cancer and prostate cancer. In yet anotherembodiment, the metastasis comprises bone metastasis. In yet anotherembodiment, the administering starts at least 6 months before theprimary surgery. In yet another embodiment, the administering starts atleast 3 months before the primary surgery. In yet another embodiment,the administering starts at least 1 month before the primary surgery. Inyet another embodiment, the administering starts within 1 week after thesurgery. In yet another embodiment, the at least one CX₃CR1 orfractalkine inhibitor comprises an agent selected from the groupconsisting of an antibody, siRNA, ribozyme, antisense, aptamer,peptidomimetic, small molecule, and combinations thereof. In yet anotherembodiment, the antibody comprises an antibody selected from apolyclonal antibody, monoclonal antibody, humanized antibody, syntheticantibody, heavy chain antibody, human antibody, a biologically activefragment of an antibody, and combinations thereof.

In one embodiment, the small molecule is selected from the groupconsisting of:

-   (i) a compound of Formula (I),

wherein in (I):

A¹ is CH₂ or cyclopentane-1,3-diyl;

n is 0, 1, 2, 3, 4 or 5 if A is CH₂, or n is 0, 1, or 2 if A iscyclopentane-1,3-diyl;

A² and A³ are both H, or A² and A³ combine to form a divalent radicalselected from the group consisting of methanediyl, ethane-1,2-diyl, orpropane-1,3-diyl;

A⁴ is CH₂, CH(CF₃), or CF₂;

R¹ is H, CN, CO₂R₆, (C₁-C₆)CH₂NH₂, or(C₁-C₆)CH₂NHC(═O)NH(4-piperidinyl);

R² and R³ are independently aryl or substituted aryl; or R² and R³combine to form a divalent fragment (a), wherein X is selected from thegroup consisting of —S—, —O—, —CH₂S—, —CH₂S(═O)—, —CH₂S(═O)₂—, —SCH₂—,—S(═O)CH₂—, —S(═O)₂CH₂—, —CH₂CH₂—, —CH═CH—, —CH₂O—, —OCH₂—,—N(CH₃)C(═O)—, and —C(═O)N(CH₃)—;

or R¹ and R² are both H, and R³ is selected from the group consistingof:

wherein ring A is selected from the group consisting of phenyl,substituted phenyl, pyridinyl, substituted pyridinyl, thiophenyl,substituted thiophenyl, 1H-pyrazole, and 1-(C₁-C₆) alkyl-1H-pyrazole;

R⁴ is nil or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl, compound ofFormula (I) is a quaternary ammonium salt;

R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl, substitutedphenyl, heteroaryl, substituted heteroaryl;

R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or substitutedaryl; and,

R⁷ is OH, CH₂OH, or C(═O)OR⁶;

-   (ii) a compound of Formula (II):

wherein:

A is a ring of formula (a), (b) or (c):

R¹ and R² independently represent H, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl or C₃-C₇ saturated or partially unsaturated cycloalkyl,

-   -   wherein in R¹ or R² the alkyl, alkenyl, alkynyl and cycloalkyl        groups are optionally and independently further substituted with        one or more substituents selected independently from the group        consisting of OH, C₁-C₆ alkoxy, CH₂OR⁴, NR⁵R⁶, CO₂R⁷ and        CONR⁸R⁹;

R³ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or C₃-C₇ saturated orpartially unsaturated cycloalkyl;

-   -   wherein in R³:        -   the alkyl, alkenyl and alkynyl chains independently and            optionally include a O, NR¹⁰ or S atom in the chain;        -   the alkyl, alkenyl, alkynyl and cycloalkyl groups are            independently and optionally further substituted by phenyl            or a 5- or 6-membered heteroaromatic ring containing 1 to 3            heteroatoms selected independently from the group consisting            of O, S and N;        -   the phenyl or heteroaromatic groups are independently and            optionally further substituted with one or more substituents            selected from the group consisting of halogen, C₁-C₄ alkyl,            OH, C₁-C₄ alkoxy, CN, CO₂R¹¹, NR¹²R¹³, C(═O)NR¹⁴R¹⁵, SO₂R¹⁶,            NR¹⁷R¹⁸ and SO₂N¹⁹R²⁰;

X is O, S or S(O);

R²¹ is H, CH₂OR²⁴, CH₂NR²⁴R²⁵, CO₂R²⁴ or C(═O)NR²⁴R²⁵;

n is 0, 1 or 2;

R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸,R¹⁹, R²⁰, R²⁴, R²⁵ and R²⁶ are independently H or C₁-C₆ alkyl;

-   (iii) a compound of Formula (VI):

wherein:

R¹ is CH₃ or CH₃CH₂;

R² is H, 2-F, 2-Cl, 3-F, 3-OCH₃, 3-CN, 3-CF₃, 3-CONH₂ or 3-SO₂CH₃;

R³ is H or CH₃;

R⁴ is H or CH₃ and

R⁵ is H; or, when R⁴ is CH₃, R⁵ is H or F;

-   (iv) a compound of Formula (VII):

wherein:

R¹ is CH₃ or CH₃CH₂;

R² is H or CH₃;

R³ is H or CH₃;

R⁴, R⁵, R⁶ and R⁷ are independently H, CH₃ or CH₂CH₃;

-   (v) a compound of Formula (VIII):

wherein:

R¹ is CH₃ or CF₃;

R² is halo, CN or C₁-C₆ alkyl;

R³ and R⁴ are independently H or CH₃;

n is 0, 1 or 2;

-   (vi) a compound of Formula (IX):

R^(A)—R^(B)   (IX),

wherein:

-   -   R^(A) is a group selected from the group consisting of:

-   -   R^(B) is a group selected from the group consisting of:

-   (vii) a compound of Formula (X):

wherein R¹ is (C₁-C₈) alkyl, substituted (C₁-C₈) alkyl, phenyl,substituted phenyl, (C₁-C₈) cycloalkyl or substituted (C₁-C₈)cycloalkyl;

-   (viii) a compound of Formula (XI):

wherein

R is selected from the group consisting of:

each occurrence of R¹ and R⁴ is independently H, (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,

R² and R³ are independently H, (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,phenyl, substituted phenyl, (C₁-C₆) cycloalkyl, substituted (C₁-C₆)cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or NHAc;

-   (ix) a compound of Formula (XII):

wherein

R is selected from the group consisting of:

each occurrence of R¹ and R4 and R⁵ is independently H, (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,

R² and R³ are independently H, (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,phenyl, substituted phenyl, (C₁-C₆) cycloalkyl, substituted (C₁-C₆)cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or NHAc;

-   (x) a compound selected from the group:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula (I) is a compound of Formula(Ia):

wherein in (Ia):

n is 0, 1, 2, 3, 4 or 5;

R¹ is H, CN, CO₂R₆, (C₁-C₆)CH₂NH₂, or(C₁-C₆)CH₂NHC(═O)NH(4-piperidinyl);

R² and R³ are independently aryl or substituted aryl; or R² and R³combine to form a divalent fragment (a), wherein X is selected from thegroup consisting of S, O, CH₂S, CH₂S(═O), CH₂S(═O)₂, SCH₂, S(═O)CH₂,S(═O)₂CH₂, CH₂CH₂, CH═CH, CH₂O, OCH₂, N(CH₃)C(═O), and C(═O)N(CH₃);

R⁴ is no atom or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl, compound(I) is a quaternary ammonium salt;

R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl, substitutedphenyl, heteroaryl, substituted heteroaryl;

R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or substitutedaryl; and,

R⁷ is OH, CH₂OH, or C(═O)OR⁶.

In one embodiment, in Formula (I) R² and R³ are independently phenyl,substituted phenyl, naphthryl or substituted naphthryl.

In one embodiment, the compound of Formula (II) is:

-   (i) a compound of Formula (III):

-   (b) a compound of Formula (IV):

-   (c) a compound of Formula (V):

or a pharmaceutically acceptable salt thereof.

In one embodiment, in Formula (X) R¹ is n-hexyl, phenyl or cyclopentyl.In another embodiment, the small molecule is selected from the groupconsisting of:5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile,N-(1-benzylpiperidin-4-yl)-9H-xanthene-9-carboxamide,5-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one,5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one, combinationsthereof, and a pharmaceutically acceptable salt thereof. In anotherembodiment, the subject is a mammal. In yet another embodiment, themammal is human. In yet another embodiment, the composition isadministered by an administration route selected from the groupconsisting of inhalational, oral, rectal, vaginal, parenteral, topical,transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, andcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a drawing illustrating the proposed model of CX₃CR1-FKNinteractions regulating the arrest of circulating cancer cells to theskeleton. The membrane-bound form of FKN, expressed on the surface ofbone marrow endothelial cells, captures CX₃CR1-expressing cancer cells.Subsequently, the soluble form of the chemokine, produced by cells ofthe bone stroma, will attract adherent cells, thus allowing theirextravasation.

FIG. 2, comprising panels A-F, illustrates the expression of CX₃CR1 inhuman breast cancer tissue arrays. Panel A illustrates a representativesample that stained negative for CX₃CR1. The majority of samplesexamined showed different degrees of positive staining for the receptorin the epithelial cells (Panels B-D; see also Table 1). Panels E-Fillustrate a negative and highly positive sample for CX₃CR1,respectively, at higher magnification. The stromal compartment staineduniformly negative for CX₃CR1. Representative images of 47 normal and202 malignant tissue cores analyzed. The BRC1502, BR722 and BR1002arrays were obtained from US Biomax (Rockville, Md.) and the stainingwas performed using an antibody against CX₃CR1 (7201) obtained fromAbcam (Cambridge, Mass.) and used at a 3.3 μg/ml concentration.(Original magnification ×100 for Panels A-D, and ×200 for Panels E-F).FIG. 2 is a set of pictures illustrating the immunohistochemistrydetection of CX₃CR1 in human tissue arrays of breast cancer. Hematoxylinand eosin staining is illustrated on the left, and immune-histochemistrydetection of CX₃CR1 in human mammary glands is illustrated on the right.Representative image of 172 malignant cores were analyzed. The BRC1502,BR722 and BR1002 arrays were obtained from US Biomax (Rockville, Md.)and the staining was performed using an antibody against CX₃CR1 (7201)obtained from Abcam (Cambridge, Mass.) and used at a 3.3 μg/mlconcentration. Original magnification was ×50.

FIG. 3 is a set of figures and a graph illustrating the role of CX₃CR1in the arrival of breast cancer cells to the skeleton. Human cancercells were directly inoculated into the blood of SCID mice and found tosignificantly differ in their ability to arrest at the bone marrow.MDA-231 cells were detected in high number when serial cryo-sections offemora or tibiae of inoculated mice were inspected by fluorescencestereomicroscopy. In contrast, similar experiments showed that MDA-436cells were detected in significantly lower numbers. When tested forCX₃CR1 expression, MDA-231 cells were found positive for this chemokinereceptor whereas MDA-436 cells failed to express it. Previously bothmembrane-bound and soluble FKN had been detected in human bone marrow;mouse marrow was also found to contain 6.4 ng/ml of FKN. Thus, theseresults show a direct correlation between CX₃CR1 expression and thearrival of cancer cells to the skeleton and suggest a role forfunctional interactions between this receptor and its ligand FKN in theprocess. The two bands observed on the western blot are most likely theresult of a previously described CX₃CR1 gene polymorphism

FIG. 4 is a graph summarizing data illustrating that both breast andprostate cancer cells are impaired in their arrival to the bone of micenull for FKN. Between 3 and 5 mice per group were used. The number oftumor cells detected is shown as mean ±SEM. (** p=0.0002, *** p=0.0001).

FIG. 5 is a fluxogram illustrating the multiple stages of the plannedresearch.

FIG. 6 is a synthetic scheme illustrating the synthesis of Compound 1(5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile).

FIG. 7 is a synthetic scheme illustrating the synthesis of Compound 2(N-(1-benzylpiperidin-4-yl)-9H-xanthene-9-carboxamide).

FIG. 8 is a set of pictures of electrophoretic gels, illustrating thefinding that Compound 1 used at 10 μM inhibits ERK phosphorylationinduced by exposure of MDA-436 breast cancer cells over-expressingCX₃CR1 to 50 nM FKN for 5 minutes.

FIG. 9 is a scheme illustrating various types of modification plannedfor Compound 1, a US28/CCR1 antagonist with inhibitory activity onCX₃CR1.

FIG. 10 is a scheme illustrating various types of modification plannedfor Compound 2, a CCR1 antagonist.

FIG. 11 is a scheme illustrating the contemplated modification of(S)—N-(3,5-bis(trifluoromethyl)benzyl)-2-(4-fluorophenyl)-4-(spiro[indene-1,4′-piperidin]-1′-yl)butanamidefor compound 1, a CCR2 receptor antagonist.

FIG. 12 illustrates the finding that red-fluorescent cancer cells firmlyadhered to green-fluorescent HBMEs after being perfused at a flow ratecorresponding to 10 dyne/cm².

FIG. 13 illustrates fluorescent cancer cells detected in the tibia of aSCID mouse, 72 hours after being inoculated into the blood circulationvia the left cardiac ventricle.

FIG. 14 is a picture illustrating bone metastatic tumors of differentsize and stage of progression produced by human fluorescent cancer cellsin SCID mice, identified by fluorescence stereomicroscopy of bonecryo-sections.

FIG. 15 is a picture illustrating the presence of fluorescent beads(shown as light circles and indicated by arrows) in the examined organsused to confirm the correct execution of cancer cell inoculation in theblood circulation on animal models.

FIGS. 16A-16B illustrate exogenous expression of wild type andfunctional mutants of the CX₃CR1 receptor in MDA-436 breast cancercells. Western blotting analysis of total cell lysates collected fromparental MDA-436 cells as well as cells stably expressing the wild typeCX₃CR1, the adhesion-impaired Y14F mutant or the chemotaxis-impairedR128N mutant of the receptor was performed. Actin was used as a loadingcontrol (FIG. 16A); the correct insertion of each exogenously expressedform of the CX₃CR1 receptor on the plasma membrane of transfectedMDA-436 cells was confirmed by cell surface protein isolation (FIG.16B). Actin detected in the cytosolic fraction was used as a loadingcontrol.

FIG. 17 is a graph illustrating sample calcium flux trace and signalcalculation.

FIGS. 18A-18C are a series of graphs illustrating calcium assay results.FIG. 18A: Calcium flux stimulated by CX3CL1 in HEK293T cells expressingthe human CX3CR1 receptor. FIG. 18B: Calcium flux stimulated by Compound3(5-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one;JMS-16) and Compound 4(5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one;JMS-17) in HEK293T cells expressing the human CX3CR1 receptor. FIG. 18C:Calcium flux stimulated by EC80 concentration (0.04 μM) of CX3CL1 incells pretreated with Compound 3 (JMS-16) and Compound 4 (JMS-17).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that metastaticdissemination may take place after primary therapeutic intervention andbe caused by cancer cells departing from either residual tumor orrecurrences. Therefore, the existing concept that the emergence ofmetastatic disease is entirely determined by events already occurredwhen surgery is performed—and that are therefore no longerpreventable—does not fully represent the actual clinical scenario. Forinstance, local surgery on the primary mammary tumor induces theproduction of a plethora of wound factors that profoundly affect theproliferation of cancer cells and could affect cells that were noteliminated by surgical excision. Prior to re-intervention, residualcancer cells in patients with positive resection margins may benefitfrom a fertile stromal environment that promotes dissemination. Thisprocess would produce secondary waves of micrometastases with at leastequal probability of developing into macroscopic tumors as those seededyears earlier. Thus, the adoption of adjuvant measures aimed tointerfere with the arrival of cancer cells to the skeleton protectscancer patients from post-surgery tumor dissemination. In oneembodiment, the cancer considered within the invention is any kind ofcancer or tumor. In another embodiment, the cancer is a solid cancer ortumor. In yet another embodiment, the cancer is breast or prostatecancer or tumor.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and peptide chemistryare those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “arrest” refers to the recruitment and/orimmobilization of circulating cancer cells to an immobilized biologicaltarget, such as an organ, for example, a bone.

A “subject”, as used therein, can be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20% or ±10%, more preferably ±5%,even more preferably ±1%, and still more preferably ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

As used herein, the term “Compound 1” refers to5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrileor a salt thereof.

As used herein, the term “Compound 2” refers toN-(1-benzylpiperidin-4-yl)-9H-xanthene-9-carboxamide or a salt thereof.

As used herein, the term “Compound 3” refers to5-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-oneor a salt thereof.

As used herein, the term “Compound 4” refers to 5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one or a saltthereof.

As used herein, a “disease” is a state of health of an animal whereinthe animal cannot maintain homeostasis, and wherein if the disease isnot ameliorated then the animal's health continues to deteriorate.

As used herein, a “disorder” in an animal is a state of health in whichthe animal is able to maintain homeostasis, but in which the animal'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compounduseful within the invention (alone or in combination with anotherpharmaceutical agent), to a subject, or application or administration ofa therapeutic agent to an isolated tissue or cell line from a subject(e.g., for diagnosis or ex vivo applications), who has cancer, a symptomof cancer or the potential to develop cancer, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectcancer, the symptoms of cancer or the potential to develop cancer. Suchtreatments may be specifically tailored or modified, based on knowledgeobtained from the field of pharmacogenomics.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.Disease and disorder are used interchangeably herein.

The terms “inhibit” and “antagonize”, as used herein, mean to reduce amolecule, a reaction, an interaction, a gene, an mRNA, and/or aprotein's expression, stability, function or activity by a measurableamount or to prevent entirely. Inhibitors are compounds that, e.g., bindto, partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate a protein, a gene,and an mRNA stability, expression, function and activity, e.g.,antagonists.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” or “pharmaceutically effective amount” of a compoundare used interchangeably to refer to the amount of the compound which issufficient to provide a beneficial effect to the subject to which thecompound is administered. The term to “treat,” as used herein, meansreducing the frequency with which symptoms are experienced by a patientor subject or administering an agent or compound to reduce the severitywith which symptoms are experienced. An appropriate therapeutic amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation. By the term “specifically bind” or“specifically binds,” as used herein, is meant that a first molecule(e.g., an antibody) preferentially binds to a second molecule (e.g., aparticular antigenic epitope), but does not necessarily bind only tothat second molecule.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof.

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound useful within the invention with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The language “pharmaceutically acceptable carrier” includes apharmaceutically acceptable salt, pharmaceutically acceptable material,composition or carrier, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting a compound(s) of the present invention within or to thesubject such that it may perform its intended function. Typically, suchcompounds are carried or transported from one organ, or portion of thebody, to another organ, or portion of the body. Each salt or carriermust be “acceptable” in the sense of being compatible with the otheringredients of the formulation, and not injurious to the subject. Someexamples of materials that may serve as pharmaceutically acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; diluent; granulating agent; lubricant; binder;disintegrating agent; wetting agent; emulsifier; coloring agent; releaseagent; coating agent; sweetening agent; flavoring agent; perfumingagent; preservative; antioxidant; plasticizer; gelling agent; thickener;hardener; setting agent; suspending agent; surfactant; humectant;carrier; stabilizer; and other non-toxic compatible substances employedin pharmaceutical formulations, or any combination thereof. As usedherein, “pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of thecompound, and are physiologically acceptable to the subject.Supplementary active compounds may also be incorporated into thecompositions.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “cycloalkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a cyclic chain hydrocarbonhaving the number of carbon atoms designated (i.e. C₃-C₆ means a cyclicgroup comprising a ring group consisting of three to six carbon atoms)and includes straight, branched chain or cyclic substituent groups.Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Most preferred is (C₃-C₆)cycloalkyl,particularly cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, the term “alkenyl,” employed alone or in combinationwith other terms, means, unless otherwise stated, a stablemono-unsaturated or di-unsaturated straight chain or branched chainhydrocarbon group having the stated number of carbon atoms. Examplesinclude vinyl, propenyl (or allyl), crotyl, isopentenyl, butadienyl,1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers. Afunctional group representing an alkene is exemplified by —CH₂—CH═CH₂.

As used herein, the term “alkynyl,” employed alone or in combinationwith other terms, means, unless otherwise stated, a stable straightchain or branched chain hydrocarbon group with a triple carbon-carbonbond, having the stated number of carbon atoms. Examples include ethynyland propynyl, and the higher homologs and isomers.

As used herein, the term “substituted alkyl,” “substituted cycloalkyl,”“substituted alkenyl” or “substituted alkynyl” means alkyl, cycloalkyl,alkenyl or alkynyl, as defined above, substituted by one, two or threesubstituents selected from the group consisting of halogen, —OH, alkoxy,—NH₂, —N(CH₃)₂, —C(═O)OH, trifluoromethyl, —C(═O)O(C₁-C₄)alkyl,—C(═O)NH₂, —C(═O)NH(C₁-C₄)alkyl, —C(═O)N((C₁-C₄)alkyl)₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂, preferably containing one or two substituentsselected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and—C(═O)OH, more preferably selected from halogen, alkoxy and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃)alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃

As used herein, the term “heteroalkenyl” by itself or in combinationwith another term means, unless otherwise stated, a stable straight orbranched chain monounsaturated or di-unsaturated hydrocarbon groupconsisting of the stated number of carbon atoms and one or twoheteroatoms selected from the group consisting of O, N, and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. Up to two heteroatomsmay be placed consecutively. Examples include —CH═CH—O—CH₃,—CH═CH—CH₂—OH, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, and —CH₂—CH═CH—CH₂—SH.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings)wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples includephenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, mostpreferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one to three carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—.The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkylfunctional group in which the aryl group is substituted. Preferred issubstituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁-C₃)alkyl”means a functional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred isheteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” meansa heteroaryl-(C₁-C₃)alkyl functional group in which the heteroaryl groupis substituted. Preferred is substituted heteroaryl-(CH₂)—.

As used herein, the term “heterocycle” or “heterocyclyl” or“heterocyclic” by itself or as part of another substituent means, unlessotherwise stated, an unsubstituted or substituted, stable, mono- ormulti-cyclic heterocyclic ring system that consists of carbon atoms andat least one heteroatom selected from the group consisting of N, O, andS, and wherein the nitrogen and sulfur heteroatoms may be optionallyoxidized, and the nitrogen atom may be optionally quaternized. Theheterocyclic system may be attached, unless otherwise stated, at anyheteroatom or carbon atom that affords a stable structure. A heterocyclemay be aromatic or non-aromatic in nature. In one embodiment, theheterocycle is a heteroaryl.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includetetrahydroquinoline and 2,3-dihydrobenzofuryl.

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin and hexamethyleneoxide.

Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl(particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl,pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl,pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles include indolyl (particularly 3-,4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl,isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benztriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

The aforementioned listing of heterocyclyl and heteroaryl moieties isintended to be representative and not limiting.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup.

For aryl, aryl-(C₁-C₃)alkyl and heterocyclyl groups, the term“substituted” as applied to the rings of these groups refers to anylevel of substitution, namely mono-, di-, tri-, tetra-, orpenta-substitution, where such substitution is permitted. Thesubstituents are independently selected, and substitution may be at anychemically accessible position. In one embodiment, the substituents varyin number between one and four. In another embodiment, the substituentsvary in number between one and three. In yet another embodiment, thesubstituents vary in number between one and two. In yet anotherembodiment, the substituents are independently selected from the groupconsisting of C₁₋₆ alkyl, —OH, C₁₋₆ alkoxy, halo, amino, acetamido andnitro. In yet another embodiment, the substituents are independentlyselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, halo,acetamido, and nitro. As used herein, where a substituent is an alkyl oralkoxy group, the carbon chain may be branched, straight or cyclic, withstraight being preferred.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies useful in the presentinvention may exist in a variety of forms including, for example,polyclonal antibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)₂, as well as single chain antibodies(scFv), camelid antibodies and humanized antibodies (Harlow et al.,1998, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

As used herein, the term “heavy chain antibody” or “heavy chainantibodies” comprises immunoglobulin molecules derived from camelidspecies, either by immunization with an antigen and subsequent isolationof sera, or by the cloning and expression of nucleic acid sequencesencoding such antibodies. The term “heavy chain antibody” or “heavychain antibodies” further encompasses immunoglobulin molecules isolatedfrom an animal with heavy chain disease, or prepared by the cloning andexpression of V_(H) (variable heavy chain immunoglobulin) genes from ananimal.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for administering the compounds and compositions of theinvention.

As used herein, “aptamer” refers to a small molecule that can bindspecifically to another molecule. Aptamers are typically eitherpolynucleotide- or peptide-based molecules. A polynucleotidal aptamer isa DNA or RNA molecule, usually comprising several strands of nucleicacids, that adopt highly specific three-dimensional conformationdesigned to have appropriate binding affinities and specificitiestowards specific target molecules, such as peptides, proteins, drugs,vitamins, among other organic and inorganic molecules. Suchpolynucleotidal aptamers can be selected from a vast population ofrandom sequences through the use of systematic evolution of ligands byexponential enrichment. A peptide aptamer is typically a loop of about10 to about 20 amino acids attached to a protein scaffold that bind tospecific ligands. Peptide aptamers may be identified and isolated fromcombinatorial libraries, using methods such as the yeast two-hybridsystem.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man is a naturally-occurring sequence.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system. As used herein, the term“exogenous” refers to any material introduced from or produced outsidean organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly five amino acids and/or sugars in size. One skilledin the art understands that generally the overall three-dimensionalstructure, rather than the specific linear sequence of the molecule, isthe main criterion of antigenic specificity and therefore distinguishesone epitope from another.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds.Synthetic polypeptides can be synthesized, for example, using anautomated polypeptide synthesizer. The term “protein” typically refersto large polypeptides. The term “peptide” typically refers to shortpolypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus. Asused herein, a “peptidomimetic” is a compound containing non-peptidicstructural elements that is capable of mimicking the biological actionof a parent peptide. A peptidomimetic may or may not comprise peptidebonds.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in a kit. The instructional material ofthe kit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container which contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Compositions

In one aspect, the composition useful in the methods of the inventioncomprises a compound of Formula (I),

wherein in (I):

A¹ is CH₂ or cyclopentane-1,3-diyl;

n is 0, 1, 2, 3, 4 or 5 if A is CH₂, or n is 0, 1, or 2 if A iscyclopentane-1,3-diyl;

A² and A³ are both H, or A² and A³ combine to form a divalent radicalselected from the group consisting of methanediyl, ethane-1,2-diyl, orpropane-1,3-diyl;

A⁴ is CH₂, CH(CF₃), or CF₂;

R¹ is H, CN, CO₂R₆, (C₁-C₆)CH₂NH₂, or(C₁-C₆)CH₂NHC(═O)NH(4-piperidinyl);

R² and R³ are independently aryl or substituted aryl; or R² and R³combine to form a divalent fragment (a), wherein X is selected from thegroup consisting of —S—, —O—, —CH₂S—, —CH₂S(═O)—, —CH₂S(═O)₂—, —SCH₂—,—S(═O)CH₂—, —S(═O)₂CH₂—, —CH₂CH₂—, —CH═CH—, —CH₂O—, —OCH₂—,—N(CH₃)C(═O)—, and —C(═O)N(CH₃)—;

or R¹ and R² are both H, and R³ is selected from the group consistingof:

wherein ring A is selected from the group consisting of phenyl,substituted phenyl, pyridinyl, substituted pyridinyl, thiophenyl,substituted thiophenyl, 1H-pyrazole, and 1-(C₁-C₆) alkyl-1H-pyrazole;

R⁴ is nil or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl, compound ofFormula (I) is a quaternary ammonium salt;

R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl, substitutedphenyl, heteroaryl, substituted heteroaryl;

R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or substitutedaryl; and,

R⁷ is OH, CH₂OH, or C(═O)OR⁶;

or a salt thereof.

In one embodiment, the composition of the invention and/or useful in themethods of the invention comprises a compound of Formula (Ia),

wherein in (Ia):

n is 0, 1, 2,3, 4 or 5;

R¹ is H, CN, CO₂R₆, (C₁-C₆)CH₂NH₂, or(C₁-C₆)CH₂NHC(═O)NH(4-piperidinyl);

R² and R³ are independently aryl or substituted aryl; or R² and R³combine to form a divalent fragment (a), wherein X is selected from thegroup consisting of S, O, CH₂S, CH₂S(═O), CH₂S(═O)₂, SCH₂, S(═O)CH₂,S(═O)₂CH₂, CH₂CH₂, CH═CH, CH₂O, OCH₂, N(CH₃)C(═O), and C(═O)N(CH₃);

R⁴ is no atom or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl, compound(I) is a quaternary ammonium salt;

R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl, substitutedphenyl, heteroaryl, substituted heteroaryl;

R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or substitutedaryl; and,

R⁷ is OH, CH₂OH, or C(═O)OR⁶;

or a salt thereof.

In one embodiment, R² and R³ are independently phenyl, substitutedphenyl, naphthryl or substituted naphthryl. In another embodiment, n is2.

In one embodiment, R⁵ is phenyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-methoxyphenyl, 3-methoxyphenyl, or 4-methoxyphenyl.

In one embodiment, in Formula (I) R¹ and R² are H, and R³ is selectedfrom the group consisting of:

In one embodiment, the compound of formula (I) is selected from thegroup consisting of:

-   5-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]    quinoxalin-4(5H)-one;-   5-(3-(4-(4-chlorophenyl)-piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one;    combinations thereof; or a salt thereof.

In another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (II),

wherein:

A is a ring of formula (a), (b) or (c):

R¹ and R² independently represent H, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl or C₃-C₇ saturated or partially unsaturated cycloalkyl,

wherein in R¹ or R² the alkyl, alkenyl, alkynyl and cycloalkyl groupsare optionally and independently further substituted with one or moresubstituents selected independently from the group consisting of OH,C₁-C₆ alkoxy, CH₂OR⁴, NR⁵R⁶, CO₂R⁷ and CONR⁸R⁹;

R³ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or C₃-C₇ saturated orpartially unsaturated cycloalkyl;

wherein in R³:

-   -   the alkyl, alkenyl and alkynyl chains independently and        optionally include a O, NR¹⁰ or S atom in the chain;    -   the alkyl, alkenyl, alkynyl and cycloalkyl groups are        independently and optionally further substituted by phenyl or a        5- or 6-membered heteroaromatic ring containing 1 to 3        heteroatoms selected independently from the group consisting of        O, S and N;    -   the phenyl or heteroaromatic groups are independently and        optionally further substituted with one or more substituents        selected from the group consisting of halogen, C₁-C₄ alkyl, OH,        C₁-C₄ alkoxy, CN, CO₂R¹¹, NR¹²R¹³, C(═O)NR¹⁴R¹⁵, SO₂R¹⁶, NR¹⁷R¹⁸        and SO₂N¹⁹R²⁰;

X is O, S or S(O);

R²¹ is H, CH₂OR²⁴, CH²NR²⁴R²⁵, CO₂R²⁴ or C(═O)NR²⁴R²⁵;

n is 0, 1 or 2;

R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸,R¹⁹, R²⁰, R²⁴, R²⁵ and R²⁶ are independently H or C₁-C₆ alkyl;

or a salt thereof.

In one embodiment, the compound of Formula (II) is a compound of Formula(III) or a salt thereof:

In one embodiment, the compound of Formula (II) is a compound of Formula(IV) or a salt thereof:

In one embodiment, the compound of Formula (II) is a compound of Formula(V) or a salt thereof:

In one embodiment, the compound of Formula (II) is selected from thegroup consisting of:

-   (2R)-2-{[2-amino-5-(benzyloxy)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}-4-methylpentan-1-ol;-   (2R)-2-({2-amino-5-[(3-methoxybenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-7-yl}amino)-4-methylpentan-1-ol;-   (2R)-2-{[2-amino-5-(2-phenylethoxy)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}-4-methylpentan-1-ol;-   (2R)-2-{[2-amino-5-(2-phenoxyethoxy)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}-4-methylpentan-1-ol;-   (2R)-2-[{2-amino-5-[(2-methylbenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-7-yl}    (methyl)amino]-4-methylpentan-1-ol;-   (2R)-2-[{2-amino-5-[(4-chlorobenzyl)oxy][1,3]thiazolo[4,5-d]pyrimlidin-7-yl}(methyl)amino]-4-methylpentan1-ol;-   (2R)-2-[[(2-amino-5-[(3-chlorobenzyl)oxy][1,3]thiazolo    4,5-d]pyrimidin-7-yl}(methyl)amino]-4-methylpentan-1-ol;-   (2R)-2-[{2-amino-5-[(2-methoxybenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-7-yl}(methyl)amino]-4-methylpentan-1-ol;-   (2R)-2-[[2-amino-5-(benzyloxy)[1,3]thiazolo[4,5-d]pyrimidin-7-yl](methyl)amino]-4-methylpentan-1-ol;-   (2R)-[{2-amino-5-[(4-bromo-2-fluorobenzyl)-(R_(S),S_(S))-sulfinyl][1,3]thiazolo[4,5-d]pyrimidin-7-yl}(methyl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[2-(4-bromophenyl)ethyl](R_(S),S_(S))-sulfinyl}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[2-(2-bromophenyl)ethyl](R_(S),S_(S))-sulfinyl}[1,3]thiazol[4,5-d]pyrimidin-7-yl)amino]4-methylpentan-1-ol;-   (R)-2-[(2-amino-5-{[2-(2-bromophenyl)ethyl](R_(S),S_(S))-sulfinyl}[1,3]thiazolo[4,5-d]pyrimidin-7    yl)(methyl)amino]-4-methylpentan-1-ol;-   2-[(2,3-difluorobenzyl)oxy]-4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}pteridin-7(8H)-one;-   4-[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-2-[(3-methoxybenzyl)oxy]pteridin-7(8H)-one;-   2-[(2-chloro-3-methoxybenzyl)oxy]-4-[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}pteridin-7(8H)one;-   4-([(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-2-(2-phenylethoxy)pteridin-7(8H)-one;-   4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-2-(2-phenoxyethoxy)pteridin-7(8H)-one;-   2-[(2-chlorobenzyl)oxy]-4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}pteridin-7(8H)-one;-   2-[(4-chlorobenzyl)oxy]-4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}pteridin-7(8H)-one;-   4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-2-[(4-methylbenzyl)oxy]pteridin-7(8H)-one;-   4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-2-[(3-methylbenzyl)oxy]pteridin-7(8H)-one;-   2-[(3-chlorobenzyl)oxy]-4-{[(1S,2S)-2-hydroxyl-(hydroxymethyl)propyl]amino}-7-oxo-7,8-dihydropteridine-6-carboxamide;-   2-[(2,3-difluorobenzyl)-(R_(S),S_(S))-sulfinyl]-4-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}pteridin-7(8H)-one;-   5-(benzyloxy)-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)one;-   7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-5-[(3-methoxybenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}-5-(2-phenylethoxy)[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-(benzyloxy)-7-{[(1R)-1-(hydroxymethyl)butyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-{[(1R)-1-(hydroxymethyl)butyl]amino}-5-{[(1S)-1-phenylethyl]oxy)[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   N-(3-{[(7-{[(1R)-1-(hydroxymethyl)butyl]amino}-2-oxo-2,3-dihydro[1,3]thiazolo[4.5-d]pyrimidin-5-yl)oxy]methyl}phenyl)-N-methylmethanesulfonamide;-   N-(3-{[(7-{[(1R)-1-(hydroxymethyl)-2-methylpropyl]amino}-2-oxo-2,3-dihydro[1,3]thiazolo[4,5-d]pyrimidin-5-yl)oxy]methyl}    phenyl)methanesulfonamide;-   5-(benzyloxy)-7-{[1-(hydroxymethyl)cyclopentyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-{[1-(hydroxymethyl)cyclopentyl]amino}-5-[(2-methylbenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-2(3H)one;-   7-{[1-(hydroxymethyl)cyclopentyl])amino}-5-[(3-methylbenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[(2-chlorobenzyl)oxy]-7-{[1-(hydroxymethyl)cyclopentyl)amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[(3-chlorobenzyl)oxy]-7-{[1-(hydroxymethyl)cyclopentyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[(4-chlorobenzyl)oxy]-7-{[1-(hydroxymethyl)cyclopentyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-{[1-(hydroxymethyl)cyclopentyl]amino)-5-[(2-methoxybenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-{[1-(hydroxymethyl)cyclopentyl]amino}-5-[(3-methoxybenzyl)oxy][1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   4-{[(7-{[1-(hydroxymethyl)cyclopentyl]amino}-2-oxo-2,3-dihydro[1,3]thiazolo[4,5-d]pyrimidin-5-yl)oxy]methyl}benzonitrile;-   (R,S)-7-[[1-(hydroxymethyl)cyclopentyl]amino]-5-(1-phenylethoxy)-thiazolo[4.5-d]pyrimidin-2(3H)-one;-   7-{[1-(hydroxymethyl)cyclopentyl]amino}-5-{[(1S)-1-phenylethyl]oxy}    [1,3]thiazolo[4.5-d]pyrimidin-2(3H)-one;-   5-{[2-(3-chlorophenyl)ethyl]-(R_(S),S_(S))-sulfinyl}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-{[2-(2-bromophenyl)ethyl]-(R_(S),S_(S))-sulfinyl}-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[(2,3-difluorobenzyl)-(R_(S),S_(S))-sulfinyl]-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[benzyl-(R_(S),S_(S))-sulfinyl]-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[(2-chlorobenzyl)-(R_(S),S_(S))-sulfinyl]-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[(4-chlorobenzyl)-(R_(S),S_(S))-sulfinyl]-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   5-[benzyl-(R_(S),S_(S))-sulfinyl]-7-{[(1R)-1-(hydroxymethyl)-2-methylpropyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   11-[3-[4-(4-Fluorophenyl)-4-hydroxy-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   6,11-Dihydro-11-[3-(4-hydroxy-4-phenyl-1-piperidinyl)-propyl]dibenzo[b,e]thiepin-11-carbonitrile;-   11-[3-[4-Hydroxy-4-(2-methylphenyl)-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   11-[3-[4-Hydroxy-4-(3-trifluoromethylphenyl)-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   11-[3-[4-Hydroxy-4-(3-trifluoromethyl-4-chlorophenyl)-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   11-[3-[4-(4-Bromophenyl)-4-hydroxy-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   11-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   6,11-Dihydro-11-[3-(4-phenyl-1-piperidinyl)propyl]-dibenzo[b,e]thiepin-11-carbonitrile;-   1-[3-(11-Cyano-6,11-dihydrodibenzo[b,e]thiepin-11-yl)-propyl]-4-phenyl-4-piperidinecarbonitrile;-   11-[3-(4-Acetyl-4-phenyl-1-piperidinyl)propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile;-   6,11-Dihydro-11-[3-[4-hydroxy-4-(phenylmethyl)-1-piperidinyl]propyl]dibenzo[b,e]thiepin-11-carbonitrile;-   6,11-Dihydro-11-[3-[4-(hydroxydiphenylmethyl)-1-piperidinyl]propyl]dibenzo[b,e]thiepin-11-carbonitrile;-   9-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-9H-xanthene-9-carbonitrile;-   5-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-carbonitrile;-   5-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-5H-dibenzo[a,d]cycloheptene-5-carbonitrile;-   11-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]oxepin-11-carbonitrile;-   11-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-6,11-dihydrodibenzo[b,e]thiepin-11-carbonitrile,    5-Oxide;-   11-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-6,11-dihydro-5-methyl-6-oxo-5H-dibenzo[b,e]azepine-11-carbonitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-(4-Fluorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-(4-Bromophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-α,α-4-triphenyl-1-piperidinepentanenitrile;-   1-(4-Cyano-4,4-diphenylbutyl)-4-phenyl-4-piperidinecarboxylic acid,    methyl ester;-   1-(4-Cyano-4,4-diphenylbutyl)-4-phenyl-4-piperidinecarboxylic acid;-   4-(Hydroxymethyl)-α,α-4-triphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-α,α-diphenyl-4-(2-thienyl)-1-piperidinepentanenitrile;-   4-Hydroxy-α,α-diphenyl-4-(2-pyridinyl)-1-piperidinepentanenitrile;-   4-Hydroxy-4-(2-naphthalenyl-α,α-diphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-r,r-diphenyl-4-(3-pyridinyl)-1-piperidinepentanenitrile;-   4-[1,1′-Biphenyl]-4-yl-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-Butyl-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-4-(4-iodophenyl)-α,α-diphenyl-1-piperidinepentanenitrile;-   4-(3-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-(3,5-Dichlorophenyl)-4-hydroxy-r,r-diphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-4-(4-methoxyphenyl-α,α-diphenyl-1-piperidinepentanenitrile;-   4-Hydroxy-α,α-diphenyl-4-[4-(trifluoromethyl)phenyl]-1-piperidinepentanenitrile;-   4-(4-Aminophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   4-[4-(Dimethylamino)phenyl]-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile;-   3-(4-Fluorophenyl)-α-(1,5-cyclohexadien-1-yl)-3-hydroxy-α-phenyl-1-piperidinehexanenitrile;-   3-(4-Fluorophenyl)-α-(1,5-cyclohexadien-1-yl)-3-hydroxy-α-phenyl-1-pyrrolidinehexanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanenitrile,    N-oxide;-   4-(4-Chlorophenyl)-4-hydroxy-1-[5-cyano-5-(1,5-cyclohexadien-1-yl)-5-phenylpentyl]-1-methyl-piperidinium;-   4-(4-Chlorophenyl)-4-hydroxy-1-[5-(1,5-cyclohexadien-1-yl)-5-phenylpentyl]-1-methyl-piperidinium;-   4-(4-Chlorophenyl)-4-hydroxy-1-[5-cyano-5-(2-naphthalenyl)pentyl]-1-methyl-piperidinium;-   4-(4-Chlorophenyl)-4-hydroxy-α-(2-naphthalenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinebutanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinehexanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidineheptanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-phenyl-1-piperidinepentanenitrile;-   α-(3-Bromo-4-methoxyphenyl)-4-(4-chlorophenyl)-4-hydroxy-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-α-(2-cyanophenyl)-4-hydroxy-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(2,3,4,5,6-pentafluorophenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(3-hydroxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-α-(2-fluorophenyl)-4-hydroxy-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinepentanenitrile;-   α-(2-Bromophenyl)-4-(4-chlorophenyl)-4-hydroxy-1-piperidinepentanenitrile,-   α,4-Bis(4-chlorophenyl)-4-hydroxy-1-piperidinepentanenitrile;-   α-(2-Chlorophenyl)-4-(4-chlorophenyl)-4-hydroxy-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-[2-(trifluoromethyl)-phenyl]-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-[2-(phenylmethoxy)-phenyl]-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-[3-(phenylmethoxy)-phenyl]-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-[4-(phenylmethoxy)-phenyl]-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(3-phenoxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(2-hydroxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(4-hydroxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(3,4,5-trimethoxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(3-nitrophenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(4-methoxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(4-ethoxyphenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(2-thienyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(3-thienyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(1-naphthalenyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(2-pyridinyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(3-pyridinyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-(1-methyl-1H-pyrrol-2-yl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-α-cyano-4-hydroxy-α-phenyl-1-piperidinepentanoic    acid, ethyl ester;-   4-(4-Chlorophenyl)-α-cyano-4-hydroxy-α-phenyl-1-piperidinepentanoic    acid, Methyl Ester;-   4-(4-Chlorophenyl)-α-(3-chloropropyl)-4-hydroxy-α-phenyl-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-methyl-α-phenyl-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-α-ethyl-4-hydroxy-α-phenyl-1-piperidinepentanenitrile;-   2-[3-[4-(4-Chlorophenyl)-4-hydroxy-1-piperidinyl]propyl]-2-phenylbutanedinitrile;-   4-(4-Chlorophenyl)-α-cyclohexyl-4-hydroxy-α-phenyl-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α-phenyl-α-(1-piperidinyl)-1-piperidinepentanenitrile;-   4-(4-Chlorophenyl)-4-hydroxy-α,α-diphenyl-1-piperidinepentanoic    acid, methyl ester;-   1-(5-Amino-4,4-diphenylpentyl)-4-(4-chlorophenyl)-4-piperidinol;-   4-(4-Chlorophenyl)-4-hydroxy-1-[5-(1,5-cyclohexadien-1-yl)-5-phenylpentyl]piperidine;    and pharmaceutically acceptable salts thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (VI):

wherein:

-   -   R¹ is CH₃ or CH₃CH₂;    -   R² is H, 2-F, 2-Cl, 3-F, 3-OCH₃, 3-CN, 3-CF₃, 3-CONH₂ or        3-SO₂CH₃;    -   R³ is H or CH₃;    -   R⁴ is H or CH₃ and    -   R⁵ is H; or, when R⁴ is CH₃, R⁵ is H or F;        or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula (VI) is selected from thegroup consisting of:

-   (2R)-2-[(2-amino-5-{[(1S)-1-(2-fluorophenyl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]pentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(2-fluorophenyl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-({2-amino-5-[(1-phenylethyl)thio][1,3]thiazolo[4,5-d]pyrimidin-7-yl}amino)-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1R)-1-phenylethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   3-{(1S)-1-[(2-amino-7-{[(1R)-1-(hydroxymethyl)butyl]    amino}[1,3]thiazolo[4,5-d]pyrimidin-5-yl)thio]ethyl}benzonitrile;-   (2R)-2-{[2-amino-5-({(1S)-1-[3-(methyl    sulfonyl)phenyl]ethyl}thio)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-phenylethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]pentan-1-ol;-   3-{(1S)-1-[(2-amino-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}    [1,3]thiazolo[4,5-d]pyrimidin-5-yl)thio]ethyl}benzonitrile;-   (2R)-2-({2-amino-5-[(1-phenylpropyl)thio][1,3]thiazolo[4,5-d]pyrimidin-7-yl}amino)-4-methylpentan-1-ol;-   3-{1-[(2-amino-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]    amino}[1,3]thiazolo[4,5-d]pyrimidin-5-yl)thio]ethyl}benzamide;-   (2R)-2-{[2-amino-5-({1-[3-(trifluoromethyl)phenyl]ethyl}thio)[1,3]thiazolo[4,5-d]pyrimidin-7-yl]amino}-4-methylpentan-1-ol;-   (2R)-2-[{2-amino-5-[(1-phenylethyl)thio][1,3]thiazolo[,5-d]pyrimidin-7-yl}(methyl)amino]-4-methylpentan-1-ol:-   (2R)-2-[(2-amino-5-{[1-(2-chlorophenyl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[1-(3-methoxyphenyl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-111ethylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-I-phenylethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-phenylethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-fluoro-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-I-(2-fluorophenyl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-fluoro-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(3-fluorophenyl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;    and pharmaceutically acceptable salts thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (VII):

wherein:

-   -   R¹ is CH₃ or CH₃CH₂;    -   R² is H or CH₃;    -   R³ is H or CH₃;    -   R⁴, R⁵, R⁶ and R⁷ are independently H, CH₃ or CH₂CH₃;        or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula (VII) is selected from thegroup consisting of:

-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(pyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(2,4-dimethylpyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(2-methylpyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(3-methylpyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(2-ethylpyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(3-ethylpyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(3,5-dimethylpyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(2,4-dimethylpyridin-2-yl)propylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(2-methylpyridin-2-yl)propylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(3-methylpyridin-2-yl)propylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[1-(3,5-dimethylpyridin-2-yl)propylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[(1S/R)-(pyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;-   7-[(R)-1-hydroxy-4-methylpentan-2-ylamino]-5-[(1R/S)-(pyridin-2-yl)ethylthio]thiazolo[4,5-d]pyrimidin-2(3H)-one;    and a pharmaceutically acceptable salt thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (VIII):

wherein:

-   -   R¹ is CH₃ or CF₃;    -   R² is halo, CN or C₁-C₆ alkyl;    -   R³ and R⁴ are independently H or CH₃;    -   n is 0, 1 or 2;        or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of Formula (VIII) is selected from thegroup consisting of:

-   (2R)-2-[(2-amino-5-{[1-(5-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-I-(5-chloropyridin-2-yl)ethyl]thio}    [1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(5-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[1-(3-chloropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(3-chloropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1R)-1-(3-chloropyridin-4-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(3-fluoropyridin-2-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]-4-methylpentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(6-chloropyridin-3-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)(methyl)amino]pentan-1-ol;-   (2R)-2-[(2-amino-5-{[(1S)-1-(6-chloropyridin-3-yl)ethyl]thio}[1,3]thiazolo[4,5-d]pyrimidin-7-yl)amino]pentan-1-ol;    and-   2-{(1S)-1-[(2-amino-7-{[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}[1,3]thiazolo[4,5-d]pyrimidin-5-yl)thio]ethyl}isonicotinonitrile;    and a pharmaceutically acceptable salt thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (IX):

R^(A)—R^(B)   (IX)

wherein:

R^(A) is a group selected from the group consisting of:

and,

R^(B) is a group selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (X):

wherein R¹ is (C₁-C₈) alkyl, substituted (C₁-C₈) alkyl, phenyl,substituted phenyl, (C₁-C₈) cycloalkyl or substituted (C₁-C₈)cycloalkyl, or a pharmaceutically acceptable salt thereof.

In one embodiment, in Formula (X) R¹ is n-hexyl, phenyl or cyclopentyl.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of (XI):

wherein

-   -   R is selected from the group consisting of:

-   -   each occurrence of R¹ and R⁴ is independently H, (C₁-C₆) alkyl,        substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)        cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,    -   R² and R³ are independently H, (C₁-C₆) alkyl, substituted        (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆) cycloalkyl,        substituted (C₁-C₆) cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or        NHAc;        or a pharmaceutically acceptable salt thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound of Formula (XII):

wherein

-   -   R is selected from the group consisting of:

-   -   each occurrence of R¹ and R4 and R⁵ is independently H, (C₁-C₆)        alkyl, substituted (C₁-C₆) alkyl, phenyl, substituted phenyl,        (C₁-C₆) cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,    -   R² and R³ are independently H, (C₁-C₆) alkyl, substituted        (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆) cycloalkyl,        substituted (C₁-C₆) cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or        NHAc;        or a pharmaceutically acceptable salt thereof.

In yet another aspect, the composition useful in the methods of theinvention comprises a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

Preparation of Compounds

The compounds useful within the methods of the invention may be preparedusing methods known to those skilled in the art.

In a non-limiting example, exemplar compounds of Formula (I) may beprepared using the methodology illustrated in Ng et al., 1999, J. Med.Chem. 42:4680-94.

In a non-limiting example, exemplar compounds of Formula (I) may beprepared using the chemistry illustrated below:

In a non-limiting example, exemplar compounds of Formula (I) may beprepared using the chemistry illustrated below. The intermediatepyrrolo[1,2-a] quinoxalin-4(5H)-one may be obtained from commercialsources or synthesized according to literature procedures (Campiani etal., 1991, Synth. Commun. 21:1567-1576; Prunier et al., 1997, J. Med.Chem. 40:1808-1819).

The starting material pyrido[3,4-e]pyrrolo[1,2-a]pyrazin-6(5H)-one maybe prepared according to the following procedure (Prunier et al., 1997,J. Med. Chem. 40:1808; Guilon, 2004, J. Med. Chem. 47:1997):

The starting material pyrido[3,4-e]pyrrolo[1,2-a]pyrazin-6(5H)-one mayalso be prepared according to the following procedure (Prunier et al.,1997, J. Med. Chem. 40:1808):

The starting material pyrrolo[1,2-a]thieno[3,2-e]pyrazin-5(4H)-one maybe prepared according to the following procedure (Rault et al., 1996, J.Med. Chem. 39:2068):

The starting material1-methyl-1H-pyrazolo[4,3-e]pyrrolo[1,2-a]pyrazin-5(4H)-one may beprepared according to the following procedure (PCT Appl. Publ. No. WO2011/074709):

The following intermediates may be prepared according to the reactionschemes outlined:

In a non-limiting example, the compound of Formula (I)5-(3-(4-(4-chlorophenyl)piperidin-1-yl)cyclopentyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one may beprepared according to the following synthetic scheme:

In a non-limiting example, the compound of Formula (I)6-(2-(4-(4-chlorophenyl)piperidin-1-yl)ethyl)benzo[b]pyrrolo[1,2-d][1,4]oxazepin-7(6H)-onemay be prepared according to the following synthetic scheme:

In a non-limiting example, the compounds of Formula (II)-(V) may beprepared using the methodology illustrated in U.S. Patent ApplicationPublication No. US 20070142386, hereby incorporated in its entirety byreference.

In a non-limiting example, the compounds of Formula (VI) may be preparedusing the methodology illustrated in U.S. Patent Application PublicationNo. US 20080214578, hereby incorporated in its entirety by reference.

In a non-limiting example, the compounds of Formula (VII) may beprepared using the methodology illustrated in International PatentApplication Publication No. WO 20091201490, hereby incorporated in itsentirety by reference.

In a non-limiting example, the compounds of Formula (VIII) may beprepared using the methodology illustrated in International PatentApplication Publication No. WO 2008039139, hereby incorporated in itsentirety by reference, and Walters et al., 2008, Bioorg. Med. Chem.Lett. 18:798-803.

In a non-limiting example, the compounds of Formula (IX) may be preparedusing the methodology illustrated in the scheme below:

In a non-limiting example, the compounds of Formula (X) may be preparedusing the methodology illustrated in the scheme below:

In a non-limiting example, the compounds of Formula (XI) may be preparedusing the methodology illustrated in the scheme below:

In a non-limiting example, the compounds of Formula (XII) may beprepared using the methodology illustrated in the scheme below:

Salts

The compounds described herein may form salts with acids, and such saltsare included in the present invention. In one embodiment, the salts arepharmaceutically acceptable salts. The term “salts” embraces additionsalts of free acids that are useful within the methods of the invention.The term “pharmaceutically acceptable salt” refers to salts that possesstoxicity profiles within a range that affords utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present invention, such as for example utility inprocess of synthesis, purification or formulation of compounds usefulwithin the methods of the invention.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric and galacturonic acid.

Compositions

The invention includes a composition comprising:

-   -   (i) a compound of formula (I)

wherein:

-   -   A¹ is CH₂ or cyclopentane-1,3-diyl;    -   n is 0, 1, 2, 3, 4 or 5 if A is CH₂, or n is 0, 1,or 2 if A is        cyclopentane-1,3-diyl;    -   A² and A³ are both H; or A² and A³ combine to form a divalent        radical selected from the group consisting of methanediyl,        ethane-1,2-diyl, or propane-1,3-diyl;    -   A⁴ is CH₂, CH(CF₃), or CF₂;    -   R¹ and R² are both H, and R³ is selected from the group        consisting of:

wherein ring A is selected from the group consisting of phenyl,substituted phenyl, pyridinyl, substituted pyridinyl, thiophenyl,substituted thiophenyl, 1H-pyrazole, and 1-(C₁-C₆) alkyl-1H-pyrazole;

-   -   R⁴ is nil or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl,        compound (I) is a quaternary ammonium salt;    -   R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl,        substituted phenyl, heteroaryl, substituted heteroaryl;    -   R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or        substituted aryl; and,    -   R⁷ is OH, CH₂OH, or C(═O)OR⁶;        or a salt thereof.    -   (ii) a compound of Formula (IX):

R^(A)—R^(B)   (IX)

wherein:

-   -   R^(A) is a group selected from the group consisting of:

-   -   R^(B) is a group selected from the group consisting of:

-   -   (iii) a compound of Formula (X):

wherein R¹ is (C₁-C₈) alkyl, substituted (C₁-C₈) alkyl, phenyl,substituted phenyl, (C₁-C₈) cycloalkyl or substituted (C₁-C₈)cycloalkyl;

-   -   (iv) a compound of Formula (XI):

wherein

-   -   R is selected from the group consisting of:

-   -   each occurrence of R¹ and R⁴ is independently H, (C₁-C₆) alkyl,        substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)        cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,    -   R² and R³ are independently H, (C₁-C₆) alkyl, substituted        (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆) cycloalkyl,        substituted (C₁-C₆) cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or        NHAc;    -   (v) a compound of Formula (XII):

wherein

-   -   R is selected from the group consisting of:

-   -   each occurrence of R¹, R⁴ and R⁵ is independently H, (C₁-C₆)        alkyl, substituted (C₁-C₆) alkyl, phenyl, substituted phenyl,        (C₁-C₆) cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,    -   R² and R³ are independently H, (C₁-C₆) alkyl, substituted        (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆) cycloalkyl,        substituted (C₁-C₆) cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or        NHAc;        or a pharmaceutically acceptable salt thereof.

Antibodies of the Invention

The invention also includes a composition comprising a CX₃CR1 antibody.The invention also includes a composition comprising a fractalkineantibody. In one embodiment, the fractalkine antibody is directed tosoluble fractalkine. In another embodiment, the fractalkine antibody isdirected to membrane associated fractalkine. In yet one embodiment, theantibody comprises an antibody selected from a polyclonal antibody, amonoclonal antibody, a humanized antibody, a synthetic antibody, a heavychain antibody, a human antibody, and a biologically active fragment ofan antibody.

It will be appreciated by one skilled in the art that an antibodycomprises any immunoglobulin molecule, whether derived from naturalsources or from recombinant sources, which is able to specifically bindto an epitope present on a target molecule.

In one aspect of the invention, CX₃CR1 is directly inhibited by anantibody that specifically binds to an epitope on CX₃CR1. In anotheraspect of the invention, the effects of CX₃CR1 are blocked by anantibody that specifically binds to an epitope on a downstream effector.In still another aspect of the invention, the effects of CX₃CR1 areblocked by an antibody that binds to an epitope of an upstream regulatorof CX₃CR1.

When the CX₃CR1 inhibitor used in the compositions and methods of theinvention is a polyclonal antibody (IgG), the antibody is generated byinoculating a suitable animal with a peptide comprising full lengthCX₃CR1 protein, or a fragment thereof, an upstream regulator, orfragments thereof. These polypeptides, or fragments thereof, may beobtained by any methods known in the art, including chemical synthesisand biological synthesis, as described elsewhere herein. In this regard,an exemplary CX₃CR1 sequence is SEQ ID NO:1. Antibodies produced in theinoculated animal which specifically bind to CX₃CR1, or fragmentsthereof, are then isolated from fluid obtained from the animal.

In one aspect of the invention, fractalkine is directly inhibited by anantibody that specifically binds to an epitope on fractalkine. Inanother aspect of the invention, the effects of fractalkine are blockedby an antibody that specifically binds to an epitope on a downstreameffector. In still another aspect of the invention, the effects offractalkine are blocked by an antibody that binds to an epitope of anupstream regulator of fractalkine.

When the fractalkine inhibitor used in the compositions and methods ofthe invention is a polyclonal antibody (IgG), the antibody is generatedby inoculating a suitable animal with a peptide comprising full lengthfractalkine protein, or a fragment thereof, an upstream regulator, orfragments thereof. These polypeptides, or fragments thereof, may beobtained by any methods known in the art, including chemical synthesisand biological synthesis, as described elsewhere herein. In this regard,an exemplary fractalkine sequence is SEQ ID NO:2. Antibodies produced inthe inoculated animal which specifically bind to fractalkine, orfragments thereof, are then isolated from fluid obtained from theanimal.

Antibodies may be generated in this manner in several non-human mammalssuch as, but not limited to goat, sheep, horse, camel, rabbit, anddonkey. Methods for generating polyclonal antibodies are well known inthe art and are described, for example in Harlow, et al. (1998, In:Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.).

Monoclonal antibodies directed against a full length CX₃CR1 orfractalkine, or fragments thereof, may be prepared using any well-knownmonoclonal antibody preparation procedures, such as those described, forexample, in Harlow et al. (1998, In: Antibodies, A Laboratory Manual,Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood,72:109-115). Human monoclonal antibodies may be prepared by the methoddescribed in U.S. Patent Publication No. 2003/0224490. Monoclonalantibodies directed against an antigen are generated from mice immunizedwith the antigen using standard procedures as referenced herein. Nucleicacid encoding the monoclonal antibody obtained using the proceduresdescribed herein may be cloned and sequenced using technology which isavailable in the art, and is described, for example, in Wright et al.(1992, Critical Rev. Immunol. 12(3,4):125-168) and the references citedtherein.

When the antibody used in the methods of the invention is a biologicallyactive antibody fragment or a synthetic antibody corresponding toantibody to a full length CX3CR1 or fractalkine, or fragments thereof,the antibody is prepared as follows: a nucleic acid encoding the desiredantibody or fragment thereof is cloned into a suitable vector. Thevector is transfected into cells suitable for the generation of largequantities of the antibody or fragment thereof. DNA encoding the desiredantibody is then expressed in the cell thereby producing the antibody.The nucleic acid encoding the desired peptide may be cloned andsequenced using technology which is available in the art, and described,for example, in Wright et al. (1992, Critical Rev. in Immunol.12(3,4):125-168) and the references cited therein. Alternatively,quantities of the desired antibody or fragment thereof may also besynthesized using chemical synthesis technology. If the amino acidsequence of the antibody is known, the desired antibody can bechemically synthesized using methods known in the art as describedelsewhere herein.

The present invention also includes the use of humanized antibodiesspecifically reactive with an epitope present on a target molecule.These antibodies are capable of binding to the target molecule. Thehumanized antibodies useful in the invention have a human framework andhave one or more complementarity determining regions (CDRs) from anantibody, typically a mouse antibody, specifically reactive with atargeted cell surface molecule.

When the antibody used in the invention is humanized, the antibody canbe generated as described in Queen et al. (U.S. Pat. No. 6,180,370),Wright et al., (1992, Critical Rev. Immunol. 12(3,4):125-168) and in thereferences cited therein, or in Gu et al. (1997, Thrombosis & Hematocyst77(4):755-759), or using other methods of generating a humanizedantibody known in the art. The method disclosed in Queen et al. isdirected in part toward designing humanized immunoglobulins that areproduced by expressing recombinant DNA segments encoding the heavy andlight chain complementarity determining regions (CDRs) from a donorimmunoglobulin capable of binding to a desired antigen, attached to DNAsegments encoding acceptor human framework regions. Generally speaking,the invention in the Queen patent has applicability toward the design ofsubstantially any humanized immunoglobulin. Queen explains that the DNAsegments will typically include an expression control DNA sequenceoperably linked to humanized immunoglobulin coding sequences, includingnaturally-associated or heterologous promoter regions. The expressioncontrol sequences can be eukaryotic promoter systems in vectors capableof transforming or transfecting eukaryotic host cells, or the expressioncontrol sequences can be prokaryotic promoter systems in vectors capableof transforming or transfecting prokaryotic host cells. Once the vectorhas been incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the introducednucleotide sequences and as desired the collection and purification ofthe humanized light chains, heavy chains, light/heavy chain dimers orintact antibodies, binding fragments or other immunoglobulin forms mayfollow (Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NewYork, (1979), which is incorporated herein by reference).

Human constant region (CDR) DNA sequences from a variety of human cellscan be isolated in accordance with well-known procedures. Preferably,the human constant region DNA sequences are isolated from immortalizedB-cells as described in International Patent Application Publication No.WO 198702671. CDRs useful in producing the antibodies of the presentinvention may be similarly derived from DNA encoding monoclonalantibodies capable of binding to the target molecule. Such humanizedantibodies may be generated using well-known methods in any convenientmammalian source capable of producing antibodies, including, but notlimited to, mice, rats, camels, llamas, rabbits, or other vertebrates.Suitable cells for constant region and framework DNA sequences and hostcells in which the antibodies are expressed and secreted, can beobtained from a number of sources, such as the American Type CultureCollection, Manassas, Va.

One of skill in the art will further appreciate that the presentinvention encompasses the use of antibodies derived from camelidspecies. That is, the present invention includes, but is not limited to,the use of antibodies derived from species of the camelid family. As iswell known in the art, camelid antibodies differ from those of mostother mammals in that they lack a light chain, and thus comprise onlyheavy chains with complete and diverse antigen binding capabilities(Hamers-Casterman et al., 1993, Nature, 363:446-448). Such heavy-chainantibodies are useful in that they are smaller than conventionalmammalian antibodies, they are more soluble than conventionalantibodies, and further demonstrate an increased stability compared tosome other antibodies. Camelid species include, but are not limited toOld World camelids, such as two-humped camels (C. bactrianus) and onehumped camels (C. dromedarius). The camelid family further comprises NewWorld camelids including, but not limited to llamas, alpacas, vicuna andguanaco. The production of polyclonal sera from camelid species issubstantively similar to the production of polyclonal sera from otheranimals such as sheep, donkeys, goats, horses, mice, chickens, rats, andthe like. The skilled artisan, when equipped with the present disclosureand the methods detailed herein, can prepare high-titers of antibodiesfrom a camelid species. As an example, the production of antibodies inmammals is detailed in such references as Harlow et al. (1998,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.).

V_(H) proteins isolated from other sources, such as animals with heavychain disease (Seligmann et al., 1979, Immunological Rev. 48:145-167,incorporated herein by reference in its entirety), are also useful inthe compositions and methods of the invention. The present inventionfurther comprises variable heavy chain immunoglobulins produced frommice and other mammals, as detailed in Ward et al. (1989, Nature341:544-546, incorporated herein by reference in its entirety). Briefly,V_(H) genes are isolated from mouse splenic preparations and expressedin E. coli. The present invention encompasses the use of such heavychain immunoglobulins in the compositions and methods detailed herein.

Antibodies useful as CX₃CR1 or fractalkine inhibitors in the inventionmay also be obtained from phage antibody libraries. To generate a phageantibody library, a cDNA library is first obtained from mRNA which isisolated from cells, e.g., the hybridoma, which express the desiredprotein to be expressed on the phage surface, e.g., the desiredantibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Bacteriophage that encode the desired antibody may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage that express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage that do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al. (1992,Critical Rev. Immunol. 12(3,4): 125-168).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al., 1991, J. Mol. Biol. 222:581-597. Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.,1995, J. Mol. Biol. 248:97-105).

Once expressed, whole antibodies, dimers derived therefrom, individuallight and heavy chains, or other forms of antibodies can be purifiedaccording to standard procedures known in the art. Such proceduresinclude, but are not limited to, ammonium sulfate precipitation, the useof affinity columns, routine column chromatography, gel electrophoresis,and the like (see, generally, R. Scopes, “Protein Purification”,Springer-Verlag, N.Y. (1982)). Substantially pure antibodies of at leastabout 90% to 95% homogeneity are preferred, and antibodies having 98% to99% or more homogeneity most preferred for pharmaceutical uses. Oncepurified, the antibodies may then be used to practice the method of theinvention, or to prepare a pharmaceutical composition useful inpracticing the method of the invention.

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassayswhich can be used include but are not limited to competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g, CurrentProtocols in Molecular Biology, (Ausubel et al., eds.), GreenePublishing Associates and Wiley-Interscience, New York (2002)).Exemplary immunoassays are described briefly below (but are not intendedto be in any way limiting).

Methods of the Invention

The invention includes a method of preventing or treating metastasis ina subject diagnosed with cancer. The method comprises the step ofadministering to the subject in need thereof an effective amount of apharmaceutical formulation comprising at least one pharmaceuticallyacceptable carrier and at least one CX₃CR1 or fractalkine antagonist.

In one embodiment, the subject is subjected to primary surgery relatedto the cancer. In another embodiment, administration of thepharmaceutical formulation takes place before, during or after theprimary surgery. In yet another embodiment, the cancer includes breastcancer or prostate cancer. In yet another embodiment, the metastasisincludes bone metastasis. In yet another embodiment, the administrationstarts at least 6 months before the primary surgery. In yet anotherembodiment, the administration starts at least 3 months before theprimary surgery. In yet another embodiment, the administration starts atleast 1 month before the primary surgery. In yet another embodiment, theadministration starts within 1 week after the surgery.

In yet one embodiment, the at least one CX₃CR1 or fractalkine inhibitorcomprises an antibody, siRNA, a ribozyme, an antisense, an aptamer, apeptidomimetic, a small molecule, or any combination thereof. In anotherembodiment, the antibody comprises an antibody selected from apolyclonal antibody, a monoclonal antibody, a humanized antibody, asynthetic antibody, a heavy chain antibody, a human antibody, and abiologically active fragment of an antibody.

In one embodiment, the small molecule is selected from the following:

-   (i) a compound of Formula (I),

wherein in (I):

-   -   A¹ is CH₂ or cyclopentane-1,3-diyl;    -   n is 0, 1, 2, 3, 4 or 5 if A is CH₂, or n is 0, 1, or 2 if A is        cyclopentane-1,3-diyl;    -   A² and A³ are both H, or A² and A³ combine to form a divalent        radical selected from the group consisting of methanediyl,        ethane-1,2-diyl, or propane-1,3-diyl;    -   A⁴ is CH₂, CH(CF₃), or CF₂;    -   R¹ is H, CN, CO₂R₆, (C₁-C₆)CH₂NH₂, or        (C₁-C₆)CH₂NHC(═O)NH(4-piperidinyl);    -   R² and R³ are independently aryl or substituted aryl; or R² and        R³ combine to form a divalent fragment (a), wherein X is        selected from the group consisting of —S—, —O—, —CH₂S—,        —CH₂S(═O)—, —CH₂S(═O)₂—, —SCH₂—, —S(═O)CH₂—, —S(═O)₂CH₂—,        —CH₂CH₂—, —CH═CH—, —CH₂O—, —OCH₂—, —N(CH₃)C(═O)—, and        —C(═O)N(CH₃)—;

or R¹ and R² are both H, and R³ is selected from the group consistingof:

wherein ring A is selected from the group consisting of phenyl,substituted phenyl, pyridinyl, substituted pyridinyl, thiophenyl,substituted thiophenyl, 1H-pyrazole, and 1-(C₁-C₆) alkyl-1H-pyrazole;

-   -   R⁴ is nil or (C₁-C₆)alkyl, wherein if R⁴ is (C₁-C₆)alkyl,        compound of Formula (I) is a quaternary ammonium salt;    -   R⁵ is (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, phenyl,        substituted phenyl, heteroaryl, substituted heteroaryl;    -   R⁶ is H, (C₁-C₆)alkyl, substituted (C₁-C₆)alkyl, aryl or        substituted aryl; and,    -   R⁷ is OH, CH₂OH, or C(═O)OR⁶;

-   (ii) a compound of Formula (II):

wherein:

-   -   A is a ring of formula (a), (b) or (c):

-   -   R¹ and R² independently represent H, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl or C₃-C₇ saturated or partially unsaturated        cycloalkyl,    -   wherein in R¹ or R² the alkyl, alkenyl, alkynyl and cycloalkyl        groups are optionally and independently further substituted with        one or more substituents selected independently from the group        consisting of OH, C₁-C₆ alkoxy, CH₂OR⁴, NR⁵R⁶, CO₂R⁷ and        CONR⁸R⁹;    -   R³ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or C₃-C₇        saturated or partially unsaturated cycloalkyl;    -   wherein in R³:        -   the alkyl, alkenyl and alkynyl chains independently and            optionally include a O, NR¹⁰ or S atom in the chain;        -   the alkyl, alkenyl, alkynyl and cycloalkyl groups are            independently and optionally further substituted by phenyl            or a 5- or 6-membered heteroaromatic ring containing 1 to 3            heteroatoms selected independently from the group consisting            of O, S and N;        -   the phenyl or heteroaromatic groups are independently and            optionally further substituted with one or more substituents            selected from the group consisting of halogen, C₁-C₄ alkyl,            OH, C₁-C₄ alkoxy, CN, CO₂R¹¹, NR¹²R¹³, C(═O)NR¹⁴R¹⁵, SO₂R¹⁶,            NR¹⁷R¹⁸ and SO₂N¹⁹R²⁰;    -   X is O, S or S(O);    -   R²¹ is H, CH₂OR²⁴, CH²NR²⁴R²⁵, CO₂R²⁴ or C(═O)NR²⁴R²⁵;    -   n is 0, 1 or 2;    -   R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷,        R¹⁸, R¹⁹, R²⁰, R²⁴, R²⁵ and R²⁶ are independently H or C₁-C₆        alkyl;

-   (iii) a compound of Formula (VI):

wherein:

-   -   R¹ is CH₃ or CH₃CH₂;    -   R² is H, 2-F, 2-Cl, 3-F, 3-OCH₃, 3-CN, 3-CF₃, 3-CONH₂ or        3-SO₂CH₃;    -   R³ is H or CH₃;    -   R⁴ is H or CH₃ and    -   R⁵ is H; or, when R⁴ is CH₃, R⁵ is H or F;

-   (iv) a compound of Formula (VII):

wherein:

-   -   R¹ is CH₃ or CH₃CH₂;    -   R² is H or CH₃;    -   R³ is H or CH₃;    -   R⁴, R⁵, R⁶ and R⁷ are independently H, CH₃ or CH₂CH₃;

-   (v) a compound of Formula (VIII):

wherein:

-   -   R¹ is CH₃ or CF₃;    -   R² is halo, CN or C₁-C₆ alkyl;    -   R³ and R⁴ are independently H or CH₃;    -   n is 0, 1 or 2;

-   (vi) a compound of Formula (IX):

R^(A)—R^(B)   (IX),

wherein:

-   -   R^(A) is a group selected from the group consisting of:

-   -   R^(B) is a group selected from the group consisting of:

-   (vii) a compound of Formula (X):

wherein R¹ is (C₁-C₈) alkyl, substituted (C₁-C₈) alkyl, phenyl,substituted phenyl, (C₁-C₈) cycloalkyl or substituted (C₁-C₈)cycloalkyl;

-   (viii) a compound of Formula (XI):

wherein

-   -   R is selected from the group consisting of:

-   -   each occurrence of R¹ and R⁴ is independently H, (C₁-C₆) alkyl,        substituted (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆)        cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,    -   R² and R³ are independently H, (C₁-C₆) alkyl, substituted        (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆) cycloalkyl,        substituted (C₁-C₆) cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or        NHAc;

-   (ix) a compound of Formula (XII):

wherein

-   -   R is selected from the group consisting of:

-   -   each occurrence of R¹ and R4 and R⁵ is independently H, (C₁-C₆)        alkyl, substituted (C₁-C₆) alkyl, phenyl, substituted phenyl,        (C₁-C₆) cycloalkyl, or substituted (C₁-C₆) cycloalkyl; and,    -   R² and R³ are independently H, (C₁-C₆) alkyl, substituted        (C₁-C₆) alkyl, phenyl, substituted phenyl, (C₁-C₆) cycloalkyl,        substituted (C₁-C₆) cycloalkyl, halo, OR⁴, N(R⁴)(R⁴), NO₂, or        NHAc;

-   (x) a compound selected from the group:

or a pharmaceutically acceptable salt thereof.

In one embodiment, in Formula (I) R² and R³ are independently phenyl,substituted phenyl, naphthryl or substituted naphthryl.

In one embodiment, the compound of Formula (II) is:

-   (a) a compound of Formula (III):

-   (b) a compound of Formula (IV):

-   (c) a compound of Formula (V):

or a pharmaceutically acceptable salt thereof.

In one embodiment, in Formula (X) R¹ is n-hexyl, phenyl or cyclopentyl.

In one embodiment, the small molecule is5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile,N-(1-benzylpiperidin-4-yl)-9H-xanthene-9-carboxamide, or apharmaceutically acceptable salt thereof.

In one embodiment, the subject is a mammal. In another embodiment, themammal is human. In yet another embodiment, the composition isadministered by an inhalational, oral, rectal, vaginal, parenteral,topical, transdermal, pulmonary, intranasal, buccal, ophthalmic,intrathecal, or intravenous route of administration.

Combination Therapies

The compounds identified using the methods described here are useful inthe methods of the invention in combination with one or more additionalcompounds useful for treating cancer. These additional compounds maycomprise compounds identified herein or compounds, e.g., commerciallyavailable compounds, known to treat, prevent, or reduce the symptoms ofcancer and/or metastasis.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Pharmaceutical Compositions and Formulations

The invention also encompasses the use of pharmaceutical compositions ofat least one compound of the invention or a salt thereof to practice themethods of the invention.

Such a pharmaceutical composition may consist of at least one compoundof the invention or a salt thereof, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise at least one compound of the invention or a salt thereof, andone or more pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The at least one compound ofthe invention may be present in the pharmaceutical composition in theform of a physiologically acceptable salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

In an embodiment, the pharmaceutical compositions useful for practicingthe method of the invention may be administered to deliver a dose ofbetween 1 ng/kg/day and 100 mg/kg/day. In another embodiment, thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for inhalational, oral, rectal,vaginal, parenteral, topical, transdermal, pulmonary, intranasal,buccal, ophthalmic, intrathecal, intravenous or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations. Theroute(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage. The unit dosage form may be for a singledaily dose or one of multiple daily doses (e.g., about 1 to 4 or moretimes per day). When multiple daily doses are used, the unit dosage formmay be the same or different for each dose.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of at least one compound ofthe invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers, which are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionsmay be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an antioxidant and a chelating agentwhich inhibit the degradation of the compound. Preferred antioxidantsfor some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid inthe preferred range of about 0.01% to 0.3% and more preferably BHT inthe range of 0.03% to 0.1% by weight by total weight of the composition.Preferably, the chelating agent is present in an amount of from 0.01% to0.5% by weight by total weight of the composition. Particularlypreferred chelating agents include edetate salts (e.g. disodium edetate)and citric acid in the weight range of about 0.01% to 0.20% and morepreferably in the range of 0.02% to 0.10% by weight by total weight ofthe composition. The chelating agent is useful for chelating metal ionsin the composition which may be detrimental to the shelf life of theformulation. While BHT and disodium edetate are the particularlypreferred antioxidant and chelating agent respectively for somecompounds, other suitable and equivalent antioxidants and chelatingagents may be substituted therefore as would be known to those skilledin the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to or after the a surgical intervention related to cancer.Further, several divided dosages, as well as staggered dosages may beadministered daily or sequentially, or the dose may be continuouslyinfused, or may be a bolus injection. Further, the dosages of thetherapeutic formulations may be proportionally increased or decreased asindicated by the exigencies of the therapeutic or prophylacticsituation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat cancer in the patient. An effective amount of the therapeuticcompound necessary to achieve a therapeutic effect may vary according tofactors such as the activity of the particular compound employed; thetime of administration; the rate of excretion of the compound; theduration of the treatment; other drugs, compounds or materials used incombination with the compound; the state of the disease or disorder,age, sex, weight, condition, general health and prior medical history ofthe patient being treated, and like factors well-known in the medicalarts. Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation. A non-limiting example of an effectivedose range for a therapeutic compound of the invention is from about0.01 and 50 mg/kg of body weight/per day. One of ordinary skill in theart would be able to study the relevant factors and make thedetermination regarding the effective amount of the therapeutic compoundwithout undue experimentation.

The compound can be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of cancer in a patient.

In one embodiment, the compositions of the invention are administered tothe patient in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the patient in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary from subjectto subject depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient will be determined by the attendingphysical taking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 7,500 mg, about 20 μg to about 7,000 mg, about40 μg to about 6,500 mg, about 80 μg to about 6,000 mg, about 100 μg toabout 5,500 mg, about 200 μg to about 5,000 mg, about 400 μg to about4,000 mg, about 800 μg to about 3,000 mg, about 1 mg to about 2,500 mg,about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mgto about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about150 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is fromabout 0.5 μg and about 5,000 mg. In some embodiments, a dose of acompound of the invention used in compositions described herein is lessthan about 5,000 mg, or less than about 4,000 mg, or less than about3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 200 mg, or less than about 50 mg. Similarly,in some embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof cancer in a patient.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in one embodiment, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating, preventing, orreducing cancer in a patient.

Routes of Administration

Routes of administration of any of the compositions of the inventioninclude inhalational, oral, nasal, rectal, parenteral, sublingual,transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal,(trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal,and (trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, a paste, a gel, toothpaste, amouthwash, a coating, an oral rinse, or an emulsion. The compositionsintended for oral use may be prepared according to any method known inthe art and such compositions may contain one or more agents selectedfrom the group consisting of inert, non-toxic pharmaceuticallyexcipients which are suitable for the manufacture of tablets. Suchexcipients include, for example an inert diluent such as lactose;granulating and disintegrating agents such as cornstarch; binding agentssuch as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide for pharmaceutically elegantand palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents; fillers;lubricants; disintegrates; or wetting agents. If desired, the tabletsmay be coated using suitable methods and coating materials such asOPADRY™ film coating systems available from Colorcon, West Point, Pa.(e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, AqueousEnteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid preparation for oral administration may be in the form ofsolutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propylpara-hydroxy benzoates or sorbic acid). Liquid formulations of apharmaceutical composition of the invention which are suitable for oraladministration may be prepared, packaged, and sold either in liquid formor in the form of a dry product intended for reconstitution with wateror another suitable vehicle prior to use.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds usefulwithin the methods of the invention, and a further layer providing forthe immediate release of one or more compounds useful within the methodsof the invention. Using a wax/pH-sensitive polymer mix, a gastricinsoluble composition may be obtained in which the active ingredient isentrapped, ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intravenous, intraperitoneal, intramuscular, intrasternal injection, andkidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see Constanza, U.S.Pat. No. 6,323,219).

In alternative embodiments, the topically active pharmaceuticalcomposition may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In another embodiment, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another aspect, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in anamount effective to affect desired changes. As used herein “amounteffective” shall mean an amount sufficient to cover the region of skinsurface where a change is desired. An active compound should be presentin the amount of from about 0.0001% to about 15% by weight volume of thecomposition. More preferable, it should be present in an amount fromabout 0.0005% to about 5% of the composition; most preferably, it shouldbe present in an amount of from about 0.001% to about 1% of thecomposition. Such compounds may be synthetically- or naturally derived.

Buccal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may contain, for example, 0.1 to20% (w/w) of the active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein. Theexamples of formulations described herein are not exhaustive and it isunderstood that the invention includes additional modifications of theseand other formulations not described herein, but which are known tothose of skill in the art.

Rectal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants, andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, and preservatives.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389,5,582,837, and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041, WO03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as tablets,capsules, gelcaps, and caplets, which are adapted for controlled-releaseare encompassed by the present invention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In a preferred embodiment of the invention, the compounds of theinvention are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials:

Unless otherwise noted, all starting materials and resins were obtainedfrom commercial suppliers and used without purification.

ERK/Akt Assay Protocol

On Day One, cells were split (1.5×10⁴ cells per well) in complete F12media (2.5×10⁵ cells per mL) into black 96 well plates. Cells wereincubated for 4-6 hours to attach. Fluid in plates was changed (SERUMFREE media ˜100 μL per well) and plates were returned to 37° C.incubator overnight.

On Day Two, TBS, BSA, 0.25% TritonX100/TBS, and 4% formaldehyde/TBS wereremoved from the refrigerator, and allowed to warm to room temperature.Fluid in plates was changed (“SF” media-90 μL per well), and platewarmer was turned on to 37° C. Compounds were diluted: for a finalstarting concentration of 10 μM: 2 uL of 10 mM stock+200 uL of SF media.Solutions were well mixed and, using 96 well round bottomed plates,serially diluted across the plate: 20 μL of previous dilution+90 μL SFmedia. Compound was added to plate (10 μL per well), and incubated onplate warmer for 5 minutes. After 5 minutes, the media was removed and50 μL per well of 4% formaldehyde/TB S were added. The system wasincubated at room temperature on shaker for 10 minutes. The fixative wasremoved, and 50 μL per well 0.25% triton X 100/TB S were added. Thesystem was incubated at room temperature on shaker for 15 minutes. PEassay buffer and 1×PE buffer were removed from refrigerator and allowedto warm to room temperature. Plates were washed 2× with 100-200 μL perwell of 1×TBS, and blotted on paper towel. 2% BSA/TBS-20 mg/mL was madefresh for each day—500 mg BSA per 25 mL TBS. 50 μl 2% BSA/TBS were addedper well, and system was incubated at room temperature on shaker for 45minutes. Each well was washed 1× 100-200 uL TBS, followed by blotting.Primary Ab was prepared by dilutions in PE assay buffer: CST pERK 1/2;1:400; and CST pAkt; 1:400. 50 μL primary Ab were added per well, andthe system was incubated at room temperature for 1-1.5 h with shaking.The system was washed 4× with 150-200 μL per well with PE wash buffer,with blotting between washes. First wash was a quick rinse, andsubsequent washes were 3-5 minutes each. Secondary Ab (PE DELFIAEu-labeled anti rabbit) was prepared, using dilution as labeled. 100 μLof PE Enhancement solution was added per well, and cover plate wasimmediately covered with another black plate. The system was incubatedfor 5-15 minutes at room temperature on shaker, and plates were read onPackard Fusion.

A plate based phos-ERK and Akt assay may be developed using DELPHIAassay methodology. With standard fluorescent detection, reagent andmicroplate interference can contribute to high background and reducedsensitivity. DELFIA uses the principle of time-resolved fluorometry toremove background interference. Lanthanide chelates possess both longfluorescence decay times and large Stoke's shifts, properties that allowdelayed signal measurement at a wavelength with little backgroundinterference. In addition, lanthanides emit a stable fluorescent signalthat exhibits a sharp emission peak and high fluorescence intensity. TheDELFIA assay principle is virtually identical to that of a standardsandwich ELISA. Analyte is first captured on a coated microplate,followed by addition of detection antibody to complete the sandwich.Unlike ELISA, DELFIA utilizes a lanthanide chelate-labeled detectionantibody, which exhibits minimal fluorescence by itself. An enhancementstep unique to DELFIA releases the lanthanide from the antibody complex,producing a new, highly fluorescent lanthanide chelate contained withina protective micelle. 4. The amplified fluorescent signal is detectedusing time-resolved fluorometry. This detection method removesnon-specific interfering fluorescent background signal and providesDELFIA superior sensitivity it is known for.

Cell Lines and Cell Cultures.

MDA-MB-231 (MDA-231) and MDA-MB-436 (MDA-436) human breast cancer cellswere purchased from ATCC (Manassas, Va.). The PC3-ML sub-line wasderived from the parental PC-3 cells as previously described (Wang &Sterns, 1991, Differntiation 48:115-25). All cells were grown in DMEMcontaining 10% fetal bovine serum (Hyclone, Logan, Utah) and 0.1%gentamicin (Invitrogen, Carlsbad, Calif.) and kept at 37° C. and 5% CO₂.For the experiments performed in vivo, cells were engineered to stablyexpress enhanced Green Fluorescent Protein (eGFP) using a lentiviralvector from America Pharma Source (Bethesda, Md.).

Transfection and Selection of Stable Cell Lines.

The cDNAs for wild type and mutant CX₃CR1 isoforms were inserted in thepEGFP-N1 vector (Clontech, Inc., Mountain View, Calif.). MDA-436 weretransfected with 3 μg of plasmid DNA using the Lipofectamine 2000transfection system according to manufacturer's instructions(Invitrogen). Stable transfected cells were selected using geneticin(Invitrogen).

Immunohistochemistry and Tissue Array Analysis.

Breast tissue microarrays (BRC1502, BR1002) were obtained from US Biomax(Rockville, Md.) and the staining for CX₃CR1 was performed as describedpreviously (Jamieson et al., 2008, Cancer Res. 68:1715-22), with anantibody against CX3CR1 (7201) obtained from Abcam (Cambridge, Mass.)and used at a 3.3 μg/ml concentration. In total, 106 tissue cores ofbreast cancer and 36 tissue cores of normal breast were examined.

Animal Models of Metastasis.

Five week-old female CB17-SCID, C57B1/6 and Balb/c mice were obtainedfrom Taconic (Germantown, N.Y.) and housed in a germ-free barrier.C57B1/6-FKN−/− transgenic mice were obtained from Schering-Plough (nowMerck-Schering Plough, Whitehouse Station, N.J.) and bred in house.Balb/c and CB17-SCID mice were used for the detection ofbone-disseminated breast cancer cells at 24 and 72 hourspost-inoculation, respectively. C57BI/6 mice were used as same-straincontrols for the C57B1/6-FKN−/− transgenic mice to detect cancer cellsdisseminated to the skeleton at 24 hours post-inoculation. At 6-8 weeksof age, mice were anesthetized with ketamine (80 mg/kg) and xylazine (10mg/kg) and inoculated in the left cardiac ventricle with human cancercells (5×10⁵ for MDA-436 and PC3-ML cells and 1×10⁵ for MDA-231 cells ina total volume of 100 ml of DMEM/F12). Blue-fluorescent polystyrenebeads (10 μm diameter, Invitrogen-Molecular Probes) were co-injected toconfirm successful delivery in the blood circulation. All experimentswere performed in accordance with NIH guidelines for the humane use ofanimals. All protocols involving the use of animals were approved by theDrexel University College of Medicine Committee for the Use and Care ofAnimals.

Tissue Preparation and Cancer Cell Detection.

Animals were sacrificed and tissues were fixed, decalcified in 0.5M EDTAif necessary and frozen in O.C.T. embedding medium (Electron MicroscopySciences, Hatfield, Pa.) as previously described (Russell et al., 2009,Oncogene 28:412-21). Serial tissue sections of 80 μm in thickness wereobtained using a Microm HM550 cryostat (Mikron, San Marcos, Ca.).Sections of each hind leg and soft-tissue organs were transferred onglass slides, stored at −20° C. and examined for cancer cells usingeither an Olympus IX70 fluorescence inverted microscope or an OlympusSZX12 fluorescence stereomicroscope. Bright field and fluorescenceimages were acquired with an Olympus DT70 CCD color camera.

Detection of Soluble FKN in Murine Bone Marrow.

Bone marrow was flushed from the hind legs of wild type C57B1/6 mice orFKN-null mice. The cellular component was removed by centrifugation at2000 r.p.m. for 10 minutes at 4° C. Soluble FKN was detected using anELISA DuoSet kit for murine FKN (R&D Systems) as previously described(Jamieson et al., 2008, Cancer Res. 68:1715-22).

In Vitro Experimental Protocol.

Cells were serum starved for 4 hours and then exposed to 50 nMrecombinant human FKN (R&D systems, Minneapolis, Minn.) for indicatedtime points.

SDS-PAGE and Western Blotting.

Cell lysates were obtained and SDS-polyacrylamide gel electrophoresisand western blot analysis were performed as previously described (Shulbyet al., 2004, Cancer Res. 64:4393-98), with few modifications. Membraneswere probed with an antibody against CX₃CR1 (0.5 μg/ml, Torrey PinesBiolabs, East Orange, N.J.) and actin (Sigma, St. Louis, Mo.) using 5%milk as a blocking reagent. Membranes were also probed with antibodiestargeting phospho-p44/42 MAPK (Thr202/Tyr204, Cell Signaling) and totalp44/42 MAPK (Cell Signaling). All primary antibody incubations wereperformed overnight at 4° C. Primary antibody binding was detected usinghorseradish peroxidase-conjugated anti-rabbit secondary antibody(Pierce, Rockford, Ill.). Chemiluminescent signals were obtained usingSuperSignal West Femto reagents (Pierce) and detected with theFluorochem 8900 imaging system and relative software (Alpha Innotech,San Leandro, Calif.).

Immunofluorescence and Confocal Microscopy.

Cells were grown on 15 mm glass coverslips and fixed with 4%formaldehyde at 4° C., not permeabilized and incubated with an antibodyagainst CX₃CR1 (Torrey Pines Biolabs) in blocker for 30 minutes and atroom temperature. A CY3-conjugated secondary antibody (JacksonImmunoresearch, West grove, Pa.) was used to detect the primary antibodyand incubated for 30 minutes at room temperature. Nuclear staining wasobtained with Hoechst 33343. Samples were imaged using a LSMS exciterconfocal system (Carl Zeiss, Usa) connected to a Axio Imager.Z1mmicroscope mounting a Plan Apochromat 20× objective with a 0.8 numericalaperture. Images were collected using a step size of 2 μm along the zaxis using the version 4.2 of the dedicated software.

CX₃CR1 Signaling in Vitro.

Cells were serum starved for 4 hours and then exposed to 50 nMrecombinant human FKN (R&D systems, Minneapolis, Minn.) for indicatedtime points.

Cell Surface CX₃CR1 Protein Isolation.

The amount of either wild-type or functional mutant forms of CX₃CR1 thatwere expressed by MDA-436 breast cancer cells at the plasma membranelevel were measured by cell surface biotinylation, using a dedicated kit(cat. #89881) obtained from Pierce (Rockford, Ill.), according to theprotocol provided by the manufacturer.

Statistics.

Statistical significance for the in vivo studies was determined using aone-tailed Student's T-test using GraphPad Prism version 3.0 for Windows(GraphPad Software, San Diego, Calif.) and data are presented as mean±standard error of the mean (S.E.M.).

Example 1 Involvement of CX₃CR1 Receptor in Cancer Metastasis

Skeletal metastases from breast adenocarcinoma are responsible for mostof the morbidity and mortality associated with this tumor and representa significant and unsolved problem for therapy. The arrival ofcirculating cancer cells to the skeleton depends first on the adhesiveinteractions with the endothelial cells lining the bone marrowsinusoids, and then the extravasation toward chemoattractant moleculesproduced by the surrounding bone stroma.

The studies described herein provide compelling evidence for theadhesive and chemotactic interactions between the CX₃CR1 receptorexpressed by breast cancer cells and fractalkine in the bone tissue.CX₃CR1 protein was detected in tissue microarrays of normal andmalignant mammary glands. Using a pre-clinical animal model ofhematogenous metastasis breast cancer cells expressing high levels ofthis receptor were shown to have higher propensity to disseminate to theskeleton. Furthermore, studies with fractalkine-null transgenic micedemonstrated that the ablation of the adhesive and chemotactic ligand ofCX₃CR1 dramatically impairs the skeletal dissemination of breast cancercells. Finally, cells that were engineered to express exogenous wildtype or functional mutants of CX₃CR1 were employed to conclusivelyconfirm the determinant role of this receptor in the arrest of breastcancer cells to the skeleton.

Direct interference with CX₃CR1-FKN functional interactions was studied.To this end, the impact exerted by the absence of FKN on cancer cellarrival to the bone was studied by using a transgenic mouse model nullfor the chemokine. When these mice received MDA-231 cells in the bloodcirculation the inspection of targeted bones showed a dramatic reductionin the number of disseminated cancer cells, which were decreased by60-70% as compared to FKN-expressing animals. To further establish therole of CX₃CR1 in this process, similar experiments were conducted usingPC3-ML prostate cancer cells, which had previously been shown to expresshigh levels of this chemokine receptor and are highly bone metastatic(Russell et al., 2009, Oncogene 28:412). As seen for MDA-231 cells, alsoPC3-ML cells were significantly impaired in their arrival to bone wheninoculated in FKN-null mice (FIG. 4).

It should be emphasized that, although the involvement of otherchemokine receptors in the dissemination of breast cancer cells has beenproposed, transgenic models null for the chemokine ligands have neverbeen used to date. In particular, the role of CXCR4 could not beinvestigated in this fashion, as mice knockout for its chemokine ligandCXCL12-SDF-1 display a lethal phenotype.

Thus, these studies represent the first pre-clinical investigation toconclusively establish the role of a chemokine receptor in themetastatic abilities of cancer cells by genetically removing thepartnering chemokine.

Taken together, these observations suggest that blocking CX₃CR1-FKNinteractions by selective and potent pharmacological compounds couldreplicate the results provided by genetic manipulation of FKN expressionin animal models and effectively interfere with the skeletaldissemination of breast cancer cells. These antagonists would be thefirst available to be tested using a preventative strategy in clinicaltrials conducted with breast cancer patients at risk for metastaticdissemination.

Despite the relevant number of studies devoted to the characterizationof CX₃CR1 signaling and the understanding of its functional role inimmunology and rheumatology processes among others, pharmacologicaltools to impair its activation and signaling have been lacking.

In one aspect, the present invention includes the identification ofnovel non-peptide, small molecule antagonists of the chemokine receptorCX₃CR1, which inhibit or reduce the arrival of circulating cancer cellsto different organs, particularly the skeleton. The strategy isillustrated in FIG. 5.

Example 2 Synthesis of Novel Antagonists of CX₃CR1

Using non-selective chemokine antagonists which bind to chemokinereceptors homologous to CX₃CR1 as starting points, analogs from threedistinct chemical series are synthesized and optimized using medicinalchemistry to generate novel and specific CX₃CR1 antagonists.

Chemokine receptors belong to class A G protein-coupled receptors (GPCR)super family (gerard & Rollins, 2001, Nat. Immunol. 2:108). Structureactivity relationships and mutagenesis studies suggest the presence of ageneric binding pocket inside the seven transmembrane bundles (Allen etal., 2007, Annu. Rev. Immunol. 25:787).

Identification of a non-peptide small molecule antagonist for CX₃CR1 isbased on a pharmacophore approach where privileged structure motifs andnon-selective ligands of homologous receptors are screened for bindingand inhibition of functional activity. The molecular interaction of thechemokine FKN and its receptor CX₃CR1 has been extensively studiedthrough mutagenesis. A cluster of basic residues and one aromaticresidue define the key hotspots important for receptor interaction(Mizoue et al., 2001, J. Biol. Chem. 276:33906).

Basic residues near the N-terminus suggest binding to an acidic residueon the receptor, presumably the conserved glutamic acid (Glu) residue inTM7 common to chemokine receptors (Rosenkilde & Schwartz, 2006, Curr.Top. Med. Chem. 6:1319). The requirement for a phenylalanine residue(Phe) suggests an engagement with an aromatic pocket in the receptor.Membrane bound FKN binds the 7-transmembrane human cytomegalovirusreceptor US28 with nanomolar affinity. US28 not only interacts with FKNbut also binds CC chemokines such as MCP-1 (CCL2) with nanomolaraffinity (Kledal et al., 1998, FEBS Lett. 441:209).

Interestingly, non-peptide small molecule ligands are known for CCR2 andUS28, which possess common pharmacophoric features such as a basic amineand an aromatic moiety, both important for receptor affinity. Based onthis evidence two initial compounds were synthesized.

Compound 1, previously reported to be an inverse agonist of US28, wassynthesized according to FIG. 6. As compound 1 was also reported tofunction as an antagonist of CCR1 compound 2, a well-know CCR1antagonist, was synthesized according to FIG. 7.

Compound 1 was then evaluated for its ability to inhibit the activationof downstream intracellular pathways by CX₃CR1 upon stimulation withsoluble FKN. In these experiments, MDA-MB 436 human breast cancer cells,engineered to over-express CX₃CR1, were treated with 50 nM FKN for 5minutes after being incubated with compound 1 for 30 minutes. FIG. 8illustrates that compound 1 was able to inhibit the activation of theMAPK pathway by blocking phosphorylation of ERKs induced by FKN.

Compound 1 represents the first small molecule CX₃CR1 antagonist, albeitweakly potent, found based on a rational pharmacophore based approachwhich holds promise for the discovery of other new leads foroptimization into a specific CX₃CR1 antagonist.

The non-peptide US28 inverse agonist, 1, and the CCR1 antagonist, 2,provide starting points as CX₃CR1 antagonists. Various analogs ofCompounds 1 and 2 are synthesized and evaluated for affinity,selectivity, and functional efficacy. The resulting structure activityrelationships will guide improvement of potency and drug-like propertiesand selectivity. Chemical synthesis provides three distinct chemicalseries for lead optimization, based on CCR1, US28, and CCR2 antagonistsor inverse agonists. The key basic amine common to most small moleculechemokine antagonists and shown to be critical in FKN affinity to CX₃CR1is retained. An aromatic motif shown to be critical in FKN and a commonfeature in small molecule chemokine antagonists is also be incorporatedinto the design. Construction of new analogs is based on a modularapproach allowing a mix and match strategy of a right hand arylpiperidine core containing a basic amine, the central core consisting ofan alkyl, cycloalkyl, or an amide bond linker, and the left handaromatic motif typically substituted with a halogen or trifluoromethylgroup.

There are numerous examples of small molecule CCR1 receptor antagonistsin the literature, which also bear the common pharmacophoric features ofan aromatic moiety and a basic amine. The US28 inverse agonist compound1 demonstrated weak CX₃CR1 antagonism and thus is the focus of furtheroptimization following the general strategy highlighted in FIG. 9.

A series of analogs includes various changes to the left hand side ofthe molecule such as substitution to the aromatic ring, linkermodifications to explore chain length and conformational constraints tothe central core, and aromatic substitution and ring size modificationsto the aryl piperidine ring on the right hand side on the molecule.

FIG. 10 highlights the strategy to modify the compound 2. Analogsinclude compounds where the 9-xanthenyl aromatic motif is modified withelectron donating, electron withdrawing, H-bond donors and acceptors,and replacement with phenylindan-like and flutroline-like privilegedbiaryl structures found in known drugs and GPCR ligands. The centralcore is modified by varying the amide linker with reverse amide andether replacements, and the ring size of the central piperidine ring isvaried to give five, six and seven member rings.

The benzyl group is modified to examine the optimum alkyl tether length,aromatic substitution, and heterocyclic replacements for the phenylring. These analogs are synthesized using standard robust chemistrymethods and where appropriate efficient parallel synthesis methods. Thismodular approach facilitates the ability to mix and match variousoptimized motifs across different chemical series in an efficient mannerto quickly optimize for potency and selectivity.

A third chemical series is also examined, adopting a strategy modifyinga CCR2 antagonist (FIG. 11). Various analogs are made to explore theeffect of ring size of the right-hand piperidine ring, aromaticsubstitution with electron withdrawing and donating groups, H-bonddonating and acceptor modification on affinity and functional activity.Central core analogs are made to evaluate the tether chain length,introduce ring conformation constraints, and vary the substitution ofthe aromatic moiety. Variations of the right hand side are made to probethe amide linkage, and evaluate various changes to the aromatic ring. Aconcise informative set of analogs are synthesized around this CCR2antagonist in order to discover a novel lead series. The currentinformation available in the literature indicates that small moleculenon-peptide chemokine antagonists tend to be non-specific, andspecificity is built in through lead optimization. The exploration ofmultiple chemical series, the first already demonstrating weak activity,provides an excellent chance to discover several chemical lead series.Lead optimization then provide potent, specific, and drug-like CX₃CR1antagonists based on strong literature precedent from programs focusedon optimizing chemokines such as CCR2 or CCR1.

Example 3 Binding Assays

The compounds synthesized for each chemical series, according to thedifferent strategies described above, are first screened for theirbinding affinity to CX₃CR1 before being optimized to identify leadseries.

MDA-436 have been recently reported to express very low levels ofCX₃CR1. MDA-436 cells engineered to exogenously express CX₃CR1 and SKBR3cells, which endogenously express the receptor, will be used. Cells areplated in 96-well plates, which will be mounted on a Multiscreen HTSvacuum manifold apparatus (Millipore). Radioactive FKN (¹²⁵I-FKN, 25μCi, 925 kBq, Perkin Elmer) is used at a concentration of 50 pM and eachnewly synthesized compound is tested at five different concentrationsstarting from 10⁻¹⁰ M and up to 10⁻⁶M, in quadruplicate. Thenonradioactive form of the chemokine is also included in each assay atthe same concentrations used for the compounds to be evaluated.Non-specific binding is considered as the binding determined in thepresence of 0.1 μM of unlabelled FKN. Each competition-binding assay isconducted at 4° C. for 3 hours. IC⁵⁰ values is determined by non-linearregression and K_(D) and Bmax values are calculated using the equations

K _(D) =IC ₅₀ −L and =B ₀(1+(K _(D) /L).

The compounds that display affinity for CX₃CR1 with a K_(i)<100 nM arethen tested for their selectivity over the CCR1, CCR2 and US28 inbinding experiments including COS-7 cells engineered to exogenouslyoverexpress each receptor as previously reported (Liang et al., 2000, J.Biol. Chem. 275:19000). Compounds with a K_(i)>10 μM for each receptortype will be moved to the next phase

Optimization studies provide structure activity relationships for thethree distinct chemical series to generate a potent and selective leadseries with good drug-like properties. Evaluation of physico-chemicalproperties of lead-compounds, such as water solubility andlipophilicity, is performed. Thus, the increase in hydrogen bonds andaddition of non-polar groups is considered while avoiding to exceed theparameters dictated by the Lipinsky rules.

The next step is the identification of compounds with favorable ADMEproperties (Absorption, Distribution, Metabolism and Excretion) andtoxicity profiles, with the ultimate goal of ruling out unsuitable drugcandidates. As a pharmacophore approach starting from non-specificchemokine receptor antagonists is used, ADME properties for some ofthese compounds have been already established or can be easilyextrapolated. Thus, if compared to high-throughput screenings ofdifferent libraries, the approach described herein significantlyfacilitates optimization in order to move from lead-likeness todrug-likeness (FIG. 5).

(a) Solubility

The compounds' solubility are evaluated for its dissolution in watercompared against a soluble solvent using LC-UV or LC-MS/MS analysis.There are several methods to determine water solubility at the lowerconcentration ranges, but if required an exact solubility limit of atest compound is determined using a more laborious HPLC or LC/MS/MSmethod with a saturating amount of solid test compound.

(b) Plasma and Microsomal Stability

Representative analogs are evaluated for their stability in the presenceof liver microsomes by incubating the test compound for 30 minutes witha microsome concentration of 0.5 mg/ml and using NADPH as cofactor. Thisanalysis provides critical information required to optimizebioavailability and understand the potential for hepatotoxicity of atest compound, which will be tested by measuring the inhibition ofCytochrome P450. Intrinsic clearance (Cl_(int)), a critical property foroptimization in human and the pre-clinical species of the animal models,is determined based on the first order elimination constant bynon-linear regression for four species; mouse, rat, dog, and human. Testcompound stability in rat, dog, monkey, and human plasma is alsodetermined by LC-MS/MS analysis.

(c) Permeability

This assay is the gold standard for evaluating the potential for oraldosing of a drug candidate. Caco-2 human colon cells are used as apolarized monolayer grown on a solid-support filter. The compound to betested is added to one side of the monolayer and the concentration ofthe compound detected on the other side will be measured by usingLC-MS/MS analysis and used as an indication of its permeability.

(d) hERG

This assay evaluates the potential cardiac toxicity of drug candidates,which is compared to know blockers of cardiac ion channels (Cisapride orTerfenadine for hERG channel) and screened by patch clamp performedusing human cardiomyocytes.

Example 4 Inhibitory Activity of Compounds on CX₃CR1-Dependent Adhesion,Migration and Signaling of Human Breast Cancer Cells In Vitro

Compounds with good drug-like properties are tested for their ability tointerfere with the functional activities of CX₃CR1. First, compoundsthat, in addition to potently binding CX₃CR1, can also disrupt itsadhesive interaction with FKN, are identified. This process occursrapidly through the establishment of a strong and stable bond with themembrane-bound form of the chemokine. The adhesion mediated by CX₃CR1does not require G-protein activation and downstream signaling; howeverthe promotion of cell migration is dependent on the ability to transduceintracellular signals. Thus, the chemotaxis of CX₃CR1 expressing cancercells towards a concentration gradient of soluble FKN is evaluated inthe presence of the newly synthesized compounds. Finally, the activationof two downstream signaling pathways such as PI3K/Akt and MAPK isdirectly evaluated by using CX₃CR1-expressing cancer cells exposed tosoluble FKN, with or without the selected drug-candidates.

(a) Adhesion

These experiments are conducted in dynamic-flow conditions, generatingshear forces that reproduce those recorded in the bone marrow sinusoids.Thus, Human Bone Marrow Endothelial cells (HBMEs) are loaded with agreen fluorescent probe (CellTracker, Invitrogen) and plated on 24×50 mmglass coverslips coated with collagen and fibronectin and grown asmonolayers (FIG. 12).

Coverslips are mounted in a parallel-plate flow chamber (RC-27N,Warner). A flow adhesion saline buffer (320 mOsm) containing 1% bovineserum albumine ise used to incubate the cell monolayers for 10 minutes.MDA-231 breast cancer cells in suspension (2×10⁵/ml) and labeled with ared-fluorescent probe is perfused through the chamber using atachometer-regulated micro-pump (Bioptechs). The flow rate used producesshear forces similar to that recorder in bone marrow microvessels invivo (Mazo et al., 10998, J. Exp. Med. 188:465) and comprised between0.1 and of 10 dyne/cm², calculated as described (Kerfoot et al., 2003,Eur. J. Immunlol. 33:729).

After allowing the cells to flow in the chamber for 5 minutes, theperfusion rate is increased to bring the shear force to 10 dynes/cm²,thus washing off loosely and non-adherent cells. Images arestream-acquired every 2 seconds using a CCD MicroMax 1300YHS digitalcamera (Roper Scientific) connected to an Olympus IX-70 microscope andmanaged by a computer equipped with the Metamorph/Metafluor software(Molecular Devices). The number of adherent cells is measured byexamining ten separate microscopic fields (Shulby et al., 2004, CancerRes. 64:4693), either in the presence or absence of each putative CX₃CR1antagonist to be tested, which is incubated with HBMEs for 30 minutesprior perfusion of the cells in the chamber.

(b) Migration and Chemotaxis

Fluorescent MDA-231 breast cancer cells are plated on the top ofFluoroBlock inserts (8 μm pore diameter, BD Biosciences) at theconcentration of 8×10⁴ per insert. The inserts are then transferred intoa 24-well plate. Each well contains serum-free culture medium with orwithout FKN (50 nM) fields (Shulby et al., 2004, Cancer Res. 64:4693).Cells are then allowed to migrate for 24 hours following their transferto the 24-well plate. The inserts are examined using a Wallac Victor 2microplate reader (Perkin Elmer) set to use bottom illumination in orderto detect exclusively the cells migrated through the membrane.

(c) Intracellular Signaling

MDA-231 and SKBR3 human breast cancer cells are exposed to 50 nM FKN fordifferent time points and in the absence or presence of the selectedcompounds to be tested. Total cell lysate is obtained andSDS-polyacrylamide gel electrophoresis and western blot analysisperformed as previously described (Russell et al., 2009, Oncogene28:412; Dolloff et al., 2005, Oncogene 24:6848). Membranes are probedwith antibodies targeting phospho-p44/42 MAPK (Thr202/Tyr204), totalp44/42 MAPK, phospho-Akt (Ser-473) and total Akt (all from CellSignaling). All primary antibody incubations are performed overnight at4° C. Primary antibody binding is detected using horseradishperoxidase-conjugated anti-rabbit secondary antibody (Pierce).Chemiluminescent signals are obtained using SuperSignal West Femtoreagents (Pierce) and detected with a Fluorochem 8900 imaging system andrelative software (Alpha Innotech).

The potency of each newly synthesized compound tested in blocking CX₃CR1functional activity is measured by calculating its IC₅₀ by constructingas dose-response curve. Only compounds displaying IC₅₀<1 μM is moved tothe next phase, in agreement with the screening criteria previouslyadopted for receptor binding affinity (K_(i)<100 nM) and selectivity(K_(i)>10 μM).

Example 5 Inhibitory Activity of Potential CX₃CR1 Antagonists on theMetastatic Potential of Breast Cancer Cells in Animal Models

Compounds are screened for selectivity using a panel of 35 differentGPCRs. The bioavailability of each compound is established and itspotential toxicity for brain tissue investigated. Pharmacokinetic (PK)studies identify loading doses and interdose plasma concentrations to beused in the subsequent animal studies. Two different animal models ofmetastatic dissemination are employed, and either immune-compromised orimmune-competent mice will be used.

(a) Selectivity

The custom-assembled platform includes 35 different GPCRs, which will beselected among non-peptide receptors (Adenosine, Dopamine, Cannabinoids,etc) and peptide receptors (Angiotensin-II, Bradykinin, Endothelin,etc). The newly synthesized antagonists of CX₃CR1 that pass the multiplescreening phases described above are analyzed and moved to the nextphase only if showing a 1000x selectivity for CX₃CR1 over any of the 35GPCRs included in the custom testing platform.

(b) Pharmacokinetics

New compounds deemed promising enough are screened in a mousepharmacokinetic assay to determine the bioavailability, plasmaconcentration, and brain/plasma exposure. This analysis provides in-lifeobservation on each mouse, plasma concentrations at each time point ofthe assay, relative bioavailability (% F) based on the AUC (area underthe IV versus PO curve) for each test compound and concentration versustime profiles. The appropriate pharmacokinetic parameters is obtainedincluding the C_(max) and T_(max) for each compound, in addition tohalf-life, clearance, and volume of distribution after IVadministration.

For this study, conscious, fasted, female CD-1 mice with a body weightbetween 20 and 35 grams are used. Three mice are used for time point, 21mice for IV dose route and 18 mice for PO dose route. Sample blood willbe collected by cardiac puncture.

In addition to further establish the drug-like properties of the newlysynthesized antagonists of CX₃CR1, this study provides crucialinformation for the execution of the experiments in the animal models ofmetastatic dissemination. For instance, for each compound the followingparameters will be identified: the loading doses to bring plasmaconcentrations to steady state that are to be used in the next steps;and the average interdose plasma concentrations to extrapolate themaintenance doses that are to be used in the next steps.

(c) Brain Toxicity

The CX₃CR1 receptor is expressed by microglia, the resident inflammatorycells of the Central Nervous System (CNS). It has been shown thataltering CX₃CR1 expression and/or functioning dysregulates microgliaresponses and induces neurotoxicity (Cardona et al., 2006, Nat.Neurosci. 9:917). Thus, penetration of the CNS by CX₃CR1 antagonistscould produce serious toxic effects. These concerns would be partiallyaddressed by comparing the structures of newly synthesized CX₃CR1antagonists with molecules with well known high-permeability for theBlood Brain Barrier (BBB), such as imipramine and baclofen. In addition,mice matched for age and weight to those used for our in vivo metastasismodels are administered with the compounds to be tested and theresulting concentrations in CNS tissue and cerebrospinal fluid measuredby LC-MS/MS analysis. A brain/plasma exposure ratio <0.1 is consideredthe minimal requisite for each CX₃CR1 antagonist to be advanced to invivo testing.

Finally, to rule out the possibility that the growth of a primary tumorand/or the presence of metastatic disease could induce systemicconditions capable of altering the BBB permeability and allowing accessof CX₃CR1 antagonists to CNS, mice are examined for macroscopicalteration of brain morphology, loss of neuronal cells and signs ofdiffuse inflammation.

(d) Dissemination of Cancer Cells Through Haematogenous Route

MDA-231 human breast cancer cells stably expressing eGFP are inoculatedin the left cardiac ventricle of immunocompromised SCID mice (CB17-SCRF,6 weeks, female, ˜35 g body weight). A suspension of 5×10⁴ fluorescentcells/100 μl is delivered using a 27-gauge needle in animals previouslyanesthetized with 100 mg/kg ketamine and 20 mg/kg xylazine (Russell etal., 2010, Cancer Res. 70:4195; Dolloff et al., 2007, Cancer Res.67:555). Different organs are collected, processed for cryosectioningand serial sections of 80 μm in thickness inspected with a fluorescencestereomicroscope. Single cancer cells in the lower femur and uppertibia, frequently bilaterally, may be consistently detected in >80% ofanimals euthanized at either 24 or 72 hours post-inoculation (FIG. 13).If animals are allowed to progress through the study, these cellsproduce macroscopic bone metastases within 4 weeks. Single cells mayalso be identified in the lungs and adrenal glands soon after theirinoculation; however, these cells eventually disappear and fail toproduce tumors, an indication that their ability to adhere andextravasate to these organs is not equaled by the potential to surviveand proliferate. Thus, this part of the study focuses mostly on theinhibitory effect of CX₃CR1 antagonists on the adhesion andextravasation of breast cancer cells to the skeleton. Since at 72 hourspost-inoculation most of the cells arrived to the bone marrow sinusoidshave extravasated into the surrounding stroma (Muller et al., 2001,Nature 410:50), this time-point is used for the following experiments.Each antagonist is administered to mice immediately before theinoculation of cancer cells and at the loading dose extrapolated by thePK experiments described herein.

(e) Statistical Power and Sample Calculation

The evaluation of reduced bone metastatic potential by CX₃CR1antagonists requires sample size calculation. A reduction of at least50% or more in number of cells disseminated to the skeleton (andpossibly other sites) is considered a significant impairment ofbone-metastatic potential.

Considering a desired power of 0.80, a significance level (α) of 0.05, adifference in population mean (δ) of 50 and a standard deviation (σ) of50, the sample size calculated for these experiments equals to 15 mice,which corresponds to 5 mice per experiments and three totalcellinoculation experiments to be conducted. If testing up to nine newlysynthesized antagonists, these experiments require 15×9=135 mice.Control experiments also require fifteen mice and this sub-aim will thenrequire 150 mice in total.

(f) Dissemination and Metastasis of Breast Cancer Cells From the MammaryFat Pad

The tumorigenic and metastatic abilities of human cancer cells must beinvestigated in immunocompromised animal models (Welch, 1997, Clin. Exp.Metastasis 15:272). However, this approach might fail to address thepossibility that in the study the immune system of the animals receivingthe cancer cells is affected by the administration of CX₃CR1 antagonist.Based on the evidence that this receptor is involved in the traffickingof leukocytes and lymphocytes, it is appropriate to establish theanti-metastatic effect of each newly synthesized antagonist both inimmune-compromised and immune-competent mice.

i. Immuno-Compromised Animals

Fluorescent MDA-231 breast cancer cells are grafted into the fourthnipple area of 8-week female SCID mice, as a suspension of 1×10⁴ cellsin 100 μl of serum-free DMEM. Animals are inspected for palpable tumorsand weighted starting from the fifth day post-inoculation. Inoculatedmice receive a dose of the antagonist for the entire duration of theexperiments, starting from the day of cellinoculation and based on theinformation on interdose plasma concentrations extrapolated from the PKanalysis performed above. After 4 weeks animals are euthanized andfemora, tibiae, lungs, adrenal glands and brain are collected. Tissuesare processed for cryosectioning and inspected for single cancer cells,small foci and macroscopic secondary tumors (FIG. 14).

ii. Immuno-Competent Animals

Fluorescent 4T-1 murine breast cancer cells are inoculated in the fourthnipple of 8-week female Balb/C mice, as a suspension of 7×10³ cells in100 μl of serum-free DMEM. The administration of CX₃CR1 antagonists andthe processing of different tissues at the end of the 4-week experimentsis conducted as described above for SCID mice.

In a second set of experiments the primary tumor produced by theinoculation of 4T-1 cells in the mammary fat pad is resected. Thisapproach is conventionally used to extend the time during which 4T-1cells can metastasize before tumor burden is reached. This procedurealso, at least partially, simulates the scenario normally observed inthe clinic after surgical excision of primary breast cancer.

Also, in these experiments the animals are treated with CX₃CR1antagonist for the entire duration of the experiments, which use overallsurvival as endpoint. Thus, animals are inoculated with cancer cells inthe mammary fat pad and the tumors are surgically removed after threeweeks. Mice are euthanized when the recurring primary mammary tumorexceeds 1.5 cm in diameter or they become moribund for diffusemetastatic disease. Different organs are collected and inspected forsecondary tumors as described above.

(g) Measurement of Metastatic Tumors

Bright-field and fluorescence images of tissue cryosections are acquiredusing a SZX12 Olympus stereomicroscope coupled to an Olympus DT70 CCDcolor camera. Digital images will be then analyzed with ImageJ softwareat (http colon double forward slash.rsb.info.nih.gov forward slash ijforward slash) and calibrated by obtaining a pixel to millimeter ratio.The largest cross-section for each metastasis is identified; itsrelative length and width (perpendicular and centered relative to oneanother) measured and total area calculated using the ellipse formula:l×w×3.14.

(h) Statistical Analyses, Power and Sample Calculation.

The same power and sample calculations described above is be used forthe experiments included herein. For each of the three sets ofexperiments 150 mice are used (15 mice ×9 antagonists in addition to 15control mice), for a total number of 450 mice. Number and size ofskeletal metastases between groups are analyzed using a two-tailedStudent's t-test. Statistical significance between multiple groups isestablished using a one-way ANOVA, followed by Tukey's MultipleComparison Test. A value of P≤0.05 is considered statisticallysignificant. Kaplan-Meyer graphs represent data on overall survival.

Single cancer cells may be detected immediately after their arrest tosecondary organs, and the skeleton in particular. This study greatlybenefits from this powerful approach. However, it is imperative tovalidate each intracardiac inoculation to confirm that the absence ofdisseminated cancer cells is not due to misinjection. To this end,cancer cells are coinjected with blue-fluorescent polystyrene beads (10μm in diameter, Invitrogen) (FIG. 15). As these beads are carried by theblood flow in the capillary bed of all organs, their absence in theexamined tissues will induce the removal of that specific animal fromthe study.

Example 6 Functional Mutants of CX₃CR1 Receptor

Experiments were conducted with MDA-436 cells stably expressing eitherthe wild-type form of CX₃CR1 or one of the two following functionalmutants of this receptor. The first mutant was generated by inducing atyrosine to phenylalanine mutation at amino acid 14 of the firstextracellular domain of CX₃CR1 (Y14F) (Fong et al., 2002, J. Biol. Chem277:19418-23). This mutant was previously characterized for its failureto firmly bind to FKN, most likely because of the inability ofphenylalanine to be sulfated, a modification that enhances the bindingto this chemokine. Although defective in capture and adhesion, CX₃CR1(Y14F) is competent in signal transduction, but with a 100-folddecreased affinity to immobilized FKN (Fong et al., 2002, J. Biol. Chem277:19418-23). The specific involvement of CX₃CR1 in extravasation wasevaluated using a second functional mutant containing an arginine toasparagine mutation at amino acid 128, which is located in the secondintracellular loop of CX₃CR1 and in the highly conserved asparticacid-arginine-tyrosine (DRY) sequence of G-protein coupled receptor.Chemoattractant properties of chemokine receptors are dependent onG-protein activation and subsequent ability to transduce downstreamsignals following stimulation by the appropriate ligand. As the DRYsequence is required for G-protein activation, the R-to-N mutation makesthe receptor incompetent of intracellular signaling and cells expressingthe CX₃CR1 (R128N) mutant do not migrate toward FKN, while showingnormal binding/adhesion to this chemokine (Haskell et al., 1999, J.Biol. Chem. 274:10053-58). The expression of wild type and mutated formsof CX₃CR1 by MDA-436 cells was verified by western blotting performed ontotal cell lysates (FIG. 16A). In addition, the insertion of each formof the receptor at the plasma membrane of transfected cells wasconfirmed by isolation of cell surface proteins obtained bybiotinylation (FIG. 16B).

Example 7 Evaluation of CX₃CR1 Antagonists on the Homing of Cancer Cellsto the Skeleton and Soft-Tissue Organs Via Haematogenous Circulation

Human breast cancer cells are inoculated in the left cardiac ventricleof immune-compromised SCID mice (CB17-SCRF, 6 weeks, female, ˜25 g bodyweight). To allow the identification of disseminated cells bymicroscopic inspection, cells are engineered to stably express enhancedGreen Fluorescent Protein (EGFP) using a lentiviral vector from AmericaPharma Source (Bethesda, Md.). Transduced cells are then enriched forEGFP expression by flow cytometry and sorting before being used for thein vivo experiment.

A suspension of 25×10⁴ fluorescent cells/100 ml is delivered using a30-gauge needle in animals previously anesthetized with 100 mg/kgketamine and 20 mg/kg xylazine. The correct execution of intracardiacinoculation is established by the appearance of fresh arterial blood inthe Luer-Lok fitting of the hypodermic needle, which indicates thesuccessful penetration of the ventricular wall. In addition,blue-fluorescent polystyrene beads (10 mm diameter, Invitrogen-MolecularProbes) are routinely co-injected with cancer cells. Their detection byfluorescence microscopy in different organs at necropsy is used toconfirm the successful inoculation in the blood circulation.

Different organs are collected and processed for cryosectioning asfollows: Tissues are fixed in 4% Formalin for 48 hours, decalcified in0.5 M EDTA if necessary and frozen in O.C.T. embedding medium (ElectronMicroscopy Sciences, Hatfield, Pa.). Serial tissue sections of 80 μm inthickness are obtained using a Microm HM550 cryostat (Mikron, SanMarcos, Calif.). Sections of each hind leg and soft-tissue organs aretransferred to glass slides, stored at −20° C. and examined for thepresence of disseminated cancer cells using an Olympus IX50 fluorescencemicroscope connected to a Nuance spectral microscopy system (CRI).

Using this approach single cancer cells are consistently detected inbone and soft-tissue organs in >90% of animals euthanized at either 24or 72 hours post-inoculation.

Control group: mice receive 100 ml of a saline solution via theintraperitoneal route (IP) 20 to 30 minutes prior to being inoculatedwith cancer cells via the intracardiac route (IC).

Antagonist group: mice receive 100 ml of a saline solution IP containingeach compound dissolved in DMSO, 20 to 30 minutes prior to beinginoculated with cancer cells. The final dose of compound is equal to 50mg/Kg and final concentration of DMSO is 5%.

Example 8 Evaluation of CX₃CR1 Antagonists on the Chemotactic Migrationof Cancer Cells

Fluorescent human cancer cells stably expressing EGFP are plated on theinserts of the BD Falcon Fluoroblock System (upper chamber). This systemprovides a platform to monitor migration of fluorescent cells using aplate reader.

The bottom of each insert is made of a light-opaque PET microporousmembrane that absorbs fluorescence within the 490-700 nm range. Theillumination of this membrane from the bottom using a plate readerallows detecting only fluorescent cells that have migrated through themembrane towards a given chemoattractant substance located in the lowerchamber of the Fluoroblock system.

Human cancer (1×10⁵) are plated in each insert and allowed to adhere tothe membrane overnight and in the absence of control or chemoattractantsolution in the lower chamber.

The migration experiment is started by adding either serum-free culturemedium as control or the same medium containing the chemokinefractalkine (1 to 50 nM) to the lower chamber of the migration system.The potential inhibitory effect of each CX₃CR1 antagonist to be screenedis tested by adding the compound (1 nM-1 mM) to the cells in the upperchamber.

The plate reader is set to 485 nm for excitation and 510 nm foremission. Emitted fluorescence is measured at time 0 as well as at 1, 3and 6 hours and subtracted of background signal acquired by measuringemitted fluorescence in the absence of cells in the upper chamber andsolution in the lower chamber.

Example 95-(3-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one—Compound3 5-(3-chloropropyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one

Pyrrolo[1,2-a]quinoxalin-4(5H)-one (500 mg; 2.71 mmol) was dissolved inanhydrous DMF (10 mL) in a 25 mL round bottomed flask. NaH (68.6 mg;2.71 mmol; 95%) was carefully added to the flask under a blanket ofnitrogen. The reaction mixture was allowed to react for 2 h at roomtemperature after which 1-bromo-3-chloropropane (511.99 mg, 3.25 mg) wasadded and the mixture was heated to 80° C. overnight. The reactionmixture was diluted with ethyl acetate and washed with H₂O, brine, driedover sodium sulfate, then filtered and concentrated. The crude mixturewas purified by flash chromatography to yield the desired5-(3-chloropropyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one and5-(3-bromopropyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one in approximately a60:40 ratio. The mixture was used in the next step without furtherpurification. [M+1]⁺=261 Chloride; [M+1]⁺=305 Bromide. ¹H NMR (CDCl₃): δ2.18-2.42 (2H); 3.49-3.62 (1H); 3.63-3.81 (1H); 4.38-4.50 (2H);6.61-6.71 (1H); 7.13-7.514 (4H); 7.65-7.80 (2H).

5-(3-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one

5-(3-chloropropyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one (100 mg; 0.38 mmol)was added to a 5 mL microwave vessel containing a magnetic stir bar.4-(4-Chlorophenyl)piperidin-4-ol (89.3 mg; 0.42 mmol), potassiumcarbonate (78.77 mg; 0.57 mmol), and 2 mL of acetonitrile were added tothe vessel. The vessel was sealed and heated to 140° C. for 1 hour,after which the solvent was evaporated and the reaction mixture waspurified by flash chromatography to yield5-(3-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one(48.5 mg; 0.11 mmol; 29%). [M+1]⁺=436.

Example 105-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one—Compound4

5-(3-chloropropyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one (100 mg; 0.38 mmol)was added to a 5 mL microwave vessel containing a magnetic stir bar.4-(4-chlorophenyl)piperidine (82.8 mg; 0.42 mmol), potassium carbonate(78.77 mg; 0.57 mmol), and 2 mL of acetonitrile were added to thevessel. The vessel was sealed and heated to 140° C. for 1 hour, afterwhich the solvent was evaporated and the reaction mixture was purifiedby flash chromatography to yield5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one(110 mg; 0.26 mmol; 70%) [M+1]⁺=420.

Example 11 Agonist and Antagonist Activity Against the Human CX3CR1Receptor Using Calcium Mobilization (Ca²⁺) Assay Material

Cells: Mammalian HEK293T cells stably expressing human CX3CR1(Multispan, Hayward, Calif.)—Host cell: HEK293T; Transfection:Expression vector containing full-length human CX3CR1 cDNA (GenBankAccession Number NM_001337) with FLAG tag sequence at N-terminus;Propagation Medium: DMEM, 10% FBS, 1 μg/mL puromycin.

Compounds were reconstituted in DMSO at 10 mM. Control agonist CX3CL1was from Peprotech (Cat #300-31). Calcium assay kit was Screen Quest™Fluo-8 No Wash kit (AAT Bioquest, Cat #36315), and instrument wasFlexStation III (Molecular Devices).

Methods

Calcium assay: Cells expressing the CX3CR1 receptor were transientlytransfected with the chimeric G protein Gαqi5 and seeded in 384-wellplates at appropriate densities and cultured overnight. Calcium assayswere conducted according to the manufacturer's protocols. The calciumdye loading buffer was added to the cells and incubated for one hour at37° C. Calcium flux was monitored for 90 seconds with compound injectedinto the wells at 20th second. In antagonist mode, carrier or compoundswere preincubated with the cells for 30 minutes before calcium fluxmeasurement with the control agonist at EC₈₀ concentration (0.04 μM).

Data analysis: Calcium assay results were expressed as “RFU” and “%Increase in RFU” (FIG. 17). Data were represented in Mean ±SEM.Dose-response curves were fitted using “Sigmoidal dose-response(variable slope)” function in Prism 4 without constraint. EC₅₀, EC₈₀ andIC₅₀ values were derived from “% Increase in RFU”.

Discussion

Control compound CX3CL1 displayed dose-dependent stimulation of calciumflux in CX3CR1-expressing cells with an expected EC₅₀ value (FIG. 18A).Compound 3 (labeled as JMS-16) showed agonist activity at the twohighest concentrations while compound 4 (labeled as JMS-17) did not haveagonist activity (FIG. 18B). Compounds 3 and 4 inhibited CX3CL1-evokedcalcium flux with IC₅₀ values of 21 μM and 11 μM, respectively (FIG.18C). The antagonist activity with Compound 3 could be due todesensitization of the receptor during pre-incubation since it hadagonist activity.

Compound 3 was shown to have a p-ERK1/2 inhibition EC₅₀ of ˜10 nM byWestern blot analysis. Compound 5 was shown to have a p-ERK1/2inhibition EC₅₀ of ˜5 nM by Western blot analysis.

The following tables summarize the raw data for the experiments.

Control Agonist

% Increase Log [CX₃CL1] M in RFU RFU −10.0 7.6 4.7 2.6 1.6 −9.6 8.3 5.12.9 1.9 −9.1 12.2 11.0 4.4 4.1 −8.6 9.6 11.8 3.3 4.0 −8.1 25.3 14.3 9.34.8 −7.7 42.3 35.6 15.5 12.4 −7.2 59.9 52.7 21.4 20.1 −6.7 59.0 61.122.2 22.7

Agonist Mode

Log % Increase in RFU RFU [Compounds] M JMS-16 JMS-17 JMS-16 JMS-17 −4.5127.9 118.0 112.4 77.7 72.3 65.1 31.7 30.7 29.0 23.0 20.7 20.2 −4.8 80.285.0 84.7 58.0 60.8 54.8 22.1 22.5 23.3 17.1 19.2 17.8 −5.1 60.1 70.364.9 59.6 61.5 52.9 16.5 20.3 18.8 17.3 16.8 17.5 −5.4 61.1 62.3 60.552.6 50.9 47.5 15.5 16.9 17.1 15.5 15.8 15.2 −5.7 57.2 61.7 60.7 57.263.4 50.6 15.2 17.8 17.1 17.2 18.7 15.9 −6.0 58.3 63.2 63.0 55.8 53.749.4 15.6 18.7 18.6 16.5 15.7 15.9 −6.3 59.0 66.2 61.9 59.0 59.9 47.416.0 18.2 19.5 17.7 17.0 14.8 −6.6 61.6 62.1 60.1 52.7 51.8 48.0 15.518.6 17.8 16.0 15.5 14.8

Antagonist Mode

Log % Increase in RFU RFU [Compounds] M JMS-16 JMS-17 JMS-16 JMS-17 −4.54.4 3.5 2.2 2.1 4.4 3.1 1.2 0.9 0.6 0.7 1.3 1.0 −4.8 27.9 23.6 26.7 11.17.0 8.3 9.5 7.6 8.5 3.5 2.3 2.7 −5.1 45.0 38.6 38.7 39.8 31.5 24.2 15.013.0 12.3 13.2 9.5 8.6 −5.4 52.1 45.7 41.1 41.9 36.0 33.2 15.5 14.4 13.513.5 11.8 10.7 −5.7 48.9 53.1 47.0 41.9 44.7 38.4 15.6 17.9 14.4 13.814.3 12.2 −6.0 58.1 54.8 53.3 43.1 44.1 41.5 18.4 18.6 18.2 14.0 14.213.2 −6.3 46.4 46.2 42.0 40.2 47.0 23.2 15.7 14.6 14.5 13.0 14.1 7.2−6.6 45.9 41.3 44.0 39.9 40.6 37.8 13.6 14.0 14.4 12.7 12.3 11.4 EC₈₀CX₃CL1 45.9 37.2 45.8 45.9 37.2 45.8 16.0 10.9 15.8 16.0 10.9 15.8

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. An isolated CX₃CR1 or fractalkine inhibitor selected from the group consisting of an antibody, siRNA, ribozyme, antisense, aptamer, peptidomimetic, and any combinations thereof.
 2. The inhibitor of claim 1, which is an antibody selected from the group consisting of a polyclonal antibody, monoclonal antibody, humanized antibody, synthetic antibody, heavy chain antibody, human antibody, biologically active fragment of an antibody, and any combinations thereof.
 3. The inhibitor of claim 1, which is formulated in a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
 5. A method of preventing or treating metastasis in a subject diagnosed with cancer, wherein the method comprises administering to the subject in need thereof an effective amount of at least one isolated CX₃CR1 or fractalkine inhibitor selected from the group consisting of an antibody, siRNA, ribozyme, antisense, aptamer, peptidomimetic, and any combinations thereof.
 6. The method of claim 5, wherein the subject is subjected to primary surgery related to the cancer.
 7. The method of claim 6, wherein administering the at least one inhibitor takes place before, during or after the primary surgery.
 8. The method of claim 5, wherein the cancer comprises a solid cancer.
 9. The method of claim 8, wherein the solid cancer is selected from the group consisting of breast cancer and prostate cancer.
 10. The method of claim 5, wherein the metastasis comprises bone metastasis.
 11. The method of claim 7, wherein the administering starts at least 6 months before the primary surgery.
 12. The method of claim 11, wherein the administering starts at least 3 months before the primary surgery.
 13. The method of claim 12, wherein the administering starts at least 1 month before the primary surgery.
 14. The method of claim 7, wherein the administering starts within 1 week after the surgery.
 15. The method of claim 5, wherein the at least one inhibitor comprises an agent selected from the group consisting of an antibody, siRNA, ribozyme, antisense, aptamer, peptidomimetic, and any combinations thereof.
 16. The method of claim 15, wherein the antibody is selected from the group consisting of a polyclonal antibody, monoclonal antibody, humanized antibody, synthetic antibody, heavy chain antibody, human antibody, biologically active fragment of an antibody, and any combinations thereof.
 17. The method of claim 5, wherein the subject is a mammal.
 18. The method of claim 17, wherein the mammal is human.
 19. The method of claim 5, wherein the at least one inhibitor is administered by an administration route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, and any combinations thereof. 